Regulation of biological events using novel compounds

ABSTRACT

Materials and methods are disclosed for regulation of biological events such as target gene transcription and growth, proliferation or differentiation of engineered cells.

BACKGROUND OF THE INVENTION

[0001] Rapamycin is a macrolide antibiotic produced by Streptomyceshygroscopicus which binds to a FK506-binding protein, FKBP, with highaffinity to form a rapamycin:FKBP complex. Reported Kd values for thatinteraction are as low as 200 pM. The rapamycin:FKBP complex binds withhigh affinity to the large cellular protein, FRAP, to form a tripartite,[FKBP:rapamycin]:[FRAP], complex. In that complex rapamycin acts as adimerizer or adapter to join FKBP to FRAP.

[0002] A number of naturally occurring FK506 binding proteins (FKBPs)are known. See e.g. Kay, 1996, Biochem. J. 314:361-385 (review).FKBP-derived domains have been incorporated in the design of chimericproteins for use in biological switches in genetically engineered cells.Such switches rely upon ligand-mediated multimerization of the proteincomponents to trigger a desired biological event. See e.g. Spencer etal, 1993, Science 262:1019-1024 and PCT/US94/01617. While the potentimmunosuppressive activity of FK506 would limit its utility as amultimerizing agent, especially in animals, dimers of FK506 (and relatedcompounds) can be made which lack such immunosuppressive activity. Suchdimers have been shown to be effective for multimerizing chimericproteins containing FKBP-derived ligand binding domains. Rapamycin, likeFK506, is also capable of multimerizing appropriately designed chimericproteins. We have previously designed biological switches usingrapamycin and various derivatives or analogs thereof (“rapalogs”) asmultimerizing agents (see WO96/41865). In the case of rapamycin itself,its significant biological activities, including potentimmunosuppressive activity, rather severely limit its use in biologicalswitches in certain applications, especially those in animals or animalcells which are sensitive to rapamycin. Improved rapalogs for suchapplications, especially rapalogs with reduced immunosuppressiveactivity, would be very desirable.

[0003] A large number of structural variants of rapamycin have beenreported, typically arising as alternative fermentation products or fromsynthetic efforts to improve the compound's therapeutic index as animmunosuppressive agent. For example, the extensive literature onanalogs, homologs, derivatives and other compounds related structurallyto rapamycin (“rapalogs”) include, among others, variants of rapamycinhaving one or more of the following modifications relative to rapamycin:demethylation, elimination or replacement of the methoxy at C7, C42and/or C29; elimination, derivatization or replacement of the hydroxy atC13, C43 and/or C28; reduction, elimination or derivatization of theketone at C14, C24 and/or C30; replacement of the 6-membered pipecolatering with a 5-membered prolyl ring; and alternative substitution on thecyclohexyl ring or replacement of the cyclohexyl ring with a substitutedcyclopentyl ring. Additional historical information is presented in thebackground sections of U.S. Pat. Nos. 5,525,610; 5,310,903 and5,362,718.

[0004] U.S. Pat. No. 5,527,907 is illustrative of the patent literature.That document discloses a series of compounds which were synthesized inan effort to make immunosuppressive rapalogs with reduced side effects.The compounds are disclosed via seven generic structural formulas, eachfollowed by extensive lists (two to five or more columns of text each)setting forth possible substituents at various positions on therapamycin ring. The document includes over 180 synthetic examples. Themany structural variants of that invention were reported to be potentimmunosuppressive agents.

SUMMARY OF THE INVENTION

[0005] This invention provides methods and materials for multimerizingchimeric proteins in genetically engineered cells using improvedrapalogs, preferably while avoiding the immunosuppressive effects ofrapamycin.

[0006] The genetically engineered cells contain one or more recombinantnucleic acid constructs encoding specialized chimeric proteins asdescribed herein. Typically a first chimeric protein contains one ormore FKBP domains which are capable of binding to an improved rapalog ofthis invention. This first chimeric protein is also referred to hereinas an “FKBP fusion protein” and further comprises at least one proteindomain heterologous to at least one of its FKBP domains. The complexformed by the binding of the FKBP fusion protein to the rapalog iscapable of binding to a second chimeric protein which contains one ormore FRB domains (the “FRB fusion protein”). The FRB fusion proteinfurther comprises at least one protein domain heterologous to at leastone of its FRB domains. In some embodiments, the FKBP fusion protein andthe FRB fusion protein are different from one another. In otherembodiments, however, the FKBP fusion protein is also an FRB fusionprotein. In those embodiments, the chimeric protein comprises one ormore FKBP domains as well as one or more FRB domains. In such cases, thefirst and second chimeric proteins may be the same protein, may bereferred to as FKBP-FRB fusion proteins and contain at least one domainheterologous to the FKBP and/or FRB domains.

[0007] The chimeric proteins may be readily designed, based onincorporation of appropriately chosen heterologous domains, such thattheir multimerization triggers one or more of a wide variety of desiredbiological responses. The nature of the biological response triggered byrapalog-mediated complexation is determined by the choice ofheterologous domains in the fusion proteins. The heterologous domainsare therefore referred to as “action” or “effector” domains. Thegenetically engineered cells for use in practicing this invention willcontain one or more recombinant nucleic acid constructs encoding thechimeric proteins, and in certain applications, will further contain oneor more accessory nucleic acid constructs, such as one or more targetgene constructs. Illustrative biological responses, applications of thesystem and types of accessory nucleic acid constructs are discussed indetail below.

[0008] A system involving related materials and methods is disclosed inWO 96/41865 (Clackson et al) and is expected to be useful in a varietyof applications including, among others, research uses and therapeuticapplications. That system involves the use of a multimerizing agentcomprising rapamycin or a rapalog of the generic formula:

[0009] wherein U is —H, —OR¹, —SR¹, —OC(O)R¹, —OC(O)NHR¹, —NHR¹,—NHC(O)R¹, —NHSO₂—R¹ or —R²; R² is a substituted aryl or allyl oralkylaryl (e.g. benzyl or substituted benzyl); V is —OR³ or (═O); W is═O, ═NR⁴═NOR⁴, ═NNHR⁴, —NHOR⁴, —NHNHR⁴, —OR⁴, —OC(O)R⁴, —OC(O)NR⁴ or —H;Y is —OR⁵, —OC(O)R⁵ or —OC(O)NHR⁵; Z is ═O, —OR⁶, —NR⁶, —H, —NC(O)R⁶,—OC(O)R⁶ or —OC(O)NR⁶; R³ is H, —R⁷, —C(O)R⁷, —C(O)NHR⁷ or C-28/C-30cyclic carbonate; and R⁴ is H or alkyl; where R¹, R⁴, R⁵, R⁶ and R⁷ areindependently selected from H, alkyl, alkylaryl or aryl, as those termsare defined in WO 96/41865. A number of rapalogs are specificallydisclosed in that document.

[0010] The subject invention is based upon a system similar to thatdisclosed in WO 96/41865, but involves the use of improved rapalogs asthe multimerizing agents. The subject invention thus provides a methodfor multimerizing chimeric proteins in cells which comprises (a)providing appropriately engineered cells containing nucleic acidconstructs for directing the expression of the desired chimericprotein(s) and any desired accessory recombinant constructs, and (b)contacting the cells with an improved rapalog or a pharmaceuticallyacceptable derivative thereof as described herein. The rapalog forms acomplex containing itself and at least two molecules of the chimericprotein(s). Improved rapalogs for use in this invention include thefollowing.

[0011] One class of improved rapalogs for use in this invention consistsof those compounds which comprise the substructure shown in Formula I:

[0012] bearing any number of a variety of substituents, and optionallyunsaturated at one or more carbon-carbon bonds unless specified to thecontrary herein, which have a substantially reduced immunosuppressiveeffect as compared with rapamycin. By a “substantially reducedimmunosuppressive effect” we mean that the rapalog has less than 0.1,preferably less than 0.01, and even more preferably, less than 0.005times the immunosuppressive effect observed or expected with anequimolar amount of rapamycin, as measured either clinically or in anappropriate in vitro or in vivo surrogate of human immunosuppressiveactivity, preferably carried out on tissues of lymphoid origin, oralternatively, that the rapalog yields an EC50 value in such an in vitroassay which is at least ten times, preferably at least 100 times andmore preferably at least 250 times larger than the EC50 value observedfor rapamycin in the same assay.

[0013] One appropriate in vitro surrogate of immunosuppression in ahuman patient is inhibition of human T cell proliferation in vitro. Thisis a conventional assay approach that may be conducted in a number ofwell known variations using various human T cells or cells lines,including among others human PBLs and Jurkat cells. A rapalog may thusbe assayed for human immunosuppressive activity and compared withrapamycin. A decrease in immunosuppressive activity relative torapamycin measured in an appropriate in vitro assay is predictive of adecrease in immunosuppressive activity in humans, relative to rapamycin.Such in vitro assays may be used to evaluate the rapalog's relativeimmunosuppressive activity.

[0014] A variety of illustrative examples of such rapalogs are disclosedherein. This class of improved rapalogs includes, among others, thosewhich bind to human FKBP12, or inhibit its rotamase activity, within anorder of magnitude of results obtained with rapamycin in anyconventional FKBP binding or rotamase assay.

[0015] Other classes of improved rapalogs for use in this invention aredefined with reference to the structure shown in Formula II:

[0016] one of R^(C7a) and R^(C7b) is H and the other is —H, halo, —R²,—OR¹, —SR¹, —OC(O)R¹ or —OC(O)NHR¹, —NHR¹, —NR¹R², —NHC(O)R¹, or—NH—SO₂—R¹ where R²=aliphatic, heteroaliphatic, aryl, heteroaryl oralkylaryl (e.g. benzyl or substituted benzyl);

[0017] R^(C30) is halo, —OR³ or (═O);

[0018] R^(C24) is ═O, ═NR⁴═NOR⁴, ═NNHR⁴, —NHOR⁴, —NHNHR⁴, —OR⁴, —OC(O)R⁴or —OC(O)NR⁴, halo or —H;

[0019] R^(C13) and R^(C28) are independently H, halo, —OR³, —OR⁵,—OC(O)R⁵, —OC(O)NHR⁵, —SR⁵, —SC(O)R⁵, —SC(O)NHR⁵, —NR⁵R^(5′) or—N(R⁵)(CO)R^(5′);

[0020] R^(C14) is ═O, —OR⁶, —NR⁶, —H, —NC(O)R⁶, —OC(O)R⁶ or —OC(O)NR⁶;

[0021] R³ is H, —R⁷, —C(O)R⁷ or —C(O)NHR⁷ or a cyclic moiety (e.g.,carbonate) bridging C28 and C30; and,

[0022] R^(C29) is H or OR¹¹ (e.g., OH or OMe);

[0023] where each substituent may be present in either stereochemicalorientation unless otherwise indicated, and where each occurrence of R¹,R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰ and R¹¹ is independently selected from H,aliphatic, heteroaliphatic, aryl and heteroaryl; and R⁸ is H, halo, —CN,═O, —OH, —NR⁹R¹⁰, OSO₂CF₃, OSO₂F, OSO₂R⁴, OCOR^(4′), OCONR^(4′)R^(5′),or OCON(OR^(4′))R^(5′).

[0024] Improved rapalogs useful in practicing this invention, includingrapalogs of Formula II, may contain substituents in any of the possiblestereoisomeric orientations, and may comprise one stereoisomersubstantially free of other stereoisomers (>90%, and preferably >95%,free from other stereoisomers on a molar basis) or may comprise amixture of stereoisomers. One class of improved rapalogs for use in thisinvention which are of particular interest are rapalogs of Formula IIwherein one or both of R^(C13) and R^(C28) is are independently H, halo,—OR³, —OR⁵, —OC(O)R⁵, —OC(O)NHR⁵, —SR⁵, —SC(O)R⁵, —SC(O)NHR⁵, —NR⁵R^(5′)or —N(R⁵)(CO)R^(5′), where each halo moiety is independently selectedfrom F, Cl, Br and I. One subset of such compounds differs in structurefrom rapamycin only at one or both of R^(C13) and R^(C28). Anothersubset of such compounds differs in structure from rapamycin at one ormore additional positions, as set forth above in connection with FormulaII or in connection with any of the other classes of improved rapalogsnoted herein. Compounds of both subsets which are of particular note arethose in which one or both of R^(C13) and R^(C28) is a halo substituent,independently selected from F, Cl, Br and I, or a substituted orunsubstituted amino moiety or acylated derivative thereof. Thesecompounds include the 13-halo rapamycins, 28-halo rapamycins, 13,28-dihalo rapamycins and related compounds in which one or more othermoities (e.g. one or both substituents at C7, for instance), in additionto the C13 and C28 substituents, differ from the correspondingmoiety(ies) in rapamycin.

[0025] Another class of improved rapalogs for use in this inventionwhich are of particular interest are rapalogs of Formula II wherein bothR^(C24) and R^(C30) are other than ═O. This class includes 24,30-tetrahydro rapamycin and mono and diethers thereof and the24,30-dihalo rapamycins. One subset of such compounds differs instructure from rapamycin only at R^(C24) and R^(C30). Another subset ofsuch compounds differs in structure from rapamycin at one or moreadditional positions (e.g. one or both substituents at C7, forinstance), as set forth above in connection with Formula II or inconnection with any of the other classes of improved rapalogs notedherein. Another class of improved rapalogs for use in this inventionwhich are of particular interest are rapalogs of Formula II whereinR^(C7a) and R^(C7b) are moieties other than a substituted orunsubstituted allyl group or a methoxy moiety. This class includesrapalogs in which one of R^(C7a) and R^(C7b) is H and the other isphenyl, di- or tri-substituted phenyl or a mono- or di-substitutedheterocyclic moiety. Illustrative examples include among others,o,p-dialkoxyphenyl substituents (e.g., o,p-dimethoxyphenyl,o-methoxy-p-ethoxyphenyl, o-ethoxy-p-methoxyphenyl, o,p-diethoxyphenyl,o,p-di (n- or iso-)propoxyphenyl, etc.), trialkoxyphenyl substituents,monosubstituted heterocycles such as methylthiophene, etc. One subset ofsuch compounds differs in structure from rapamycin only at R^(C24) andR^(C30). Another subset of such compounds differs in structure fromrapamycin at one or more additional position, as set forth above inconnection with Formula II or in connection with any of the otherclasses of improved rapalogs noted herein.

[0026] Another class of improved rapalogs for use in this inventionwhich are of particular interest are rapalogs of Formula II wherein nis 1. This class of rapalogs includes rapalogs comprising a prolyl ringsystem in place of a pipicolate ring system. One subset of suchcompounds differs in structure from rapamycin only with respect to thepipicolate ring system. Another subset of such compounds differs instructure from rapamycin with respect to one or more additionalstructural features (e.g. one or both substituents at C7, for instance),as set forth above in connection with Formula II or in connection withany of the other classes of improved rapalogs noted herein.

[0027] Another class of improved rapalogs for use in this inventionwhich are of particular interest are rapalogs of Formula II whereinmoiety “a” is other than

[0028] One subset of such compounds differs in structure from rapamycinonly with respect to the ring system, “a”. Another subset of suchcompounds differs in structure from rapamycin with respect to one ormore additional structural features (e.g. one or both substituents atC7, for instance), as set forth above in connection with Formula II orin connection with any of the other classes of improved rapalogs notedherein. This class of rapalogs include the class of 43-epi-rapalogs inwhich the hydroxyl moiety at position 43 has the opposite stereochemicalorientation with that shown immediately above, is a mixture ofstereoisomers of the 43-hydroxyl group or contains derivatives of any ofthe foregoing, including ethers, esters, carbamates, halides and otherderivatives of any of the foregoing position 43 rapalogs. This classfurther includes rapalogs in which the cyclohexyl ring is otherwisesubstituted and/or contains 5 ring atoms in place of the characteristicsubstituted cyclohexyl ring of rapamycin. Again, the improved rapalogsas described herein are used in a method for multimerizing chimericproteins in genetically engineered cells. The method involves (a)providing appropriately engineered cells containing nucleic acidconstructs for directing the expression of the desired chimeric proteins(and any desired accessory recombinant constructs), and (b) contactingthe cells with an improved rapalog or a pharmaceutically acceptablederivative thereof.

[0029] In one embodiment, at least one of the chimeric proteins containsat least one FKBP domain whose peptide sequence differs from a naturallyoccurring FKBP peptide sequence, e.g. the peptide sequence of humanFKBP12, at up to ten amino acid residues in the peptide sequence.Preferably the number of changes in peptide sequence is limited to five,and more preferably to 1, 2, or 3. In embodiments in which the rapalogcomprises a structural modification relative to rapamycin at R^(C28), atR^(C24) and R^(C30), and/or at R^(C7a) and/or R^(C7b), it is also ofspecial interest that at least one of the chimeric proteins contains atleast one FKBP domain comprising at least one amino acid replacementrelative to the sequence of a naturally occurring FKBP, especially amammalian FKBP such as human FKBP12. Mutations of particular interestinclude replacement of either or both of Phe36 and Phe99 of human FKBP12sequence with independently selected replacement amino acids, e.g.valine, methionine, alanine or serine.

[0030] In another embodiment, at least one of the chimeric proteinscontains at least one FRB domain whose peptide sequence differs from anaturally occurring FRB peptide sequence, e.g. the FRB domain of humanFRAP, at up to ten amino acid residues in the peptide sequence.Preferably the number of changes in peptide sequence is limited to five,and more preferably to 1, 2, or 3. in many cases it will be preferredthat the FRB domain contains a single amino acid replacement relative tothe peptide sequence of the corresponding FRB domain of human FRAP orsome other mammalian FRAP/TOR species. Mutations of particular interestinclude replacement of one or more of T2098, D2102, Y2038, F2039, K2095of an FRB domain derived from human FRAP with independently selectedreplacement amino acids, e.g. A, N, H, L, or S. Also of interest are thereplacement of one or more of F1975, F1976, D2039 and N2035 of an FRBdomain derived from yeast TOR1, or the replacement of one or more ofF1978, F1979, D2042 and N2038 of an FRB domain derived from yeast TOR2,with independently selected replacement amino acids, e.g. H, L, S, A orV.

[0031] In certain embodiments the chimeric protein(s) contain at leastone modification in peptide sequence, preferably up to threemodifications, relative to naturally occurring sequences, in both one ormore FKBP domains and one or more FRB domains.

[0032] As mentioned previously, in the various embodiments of thisinvention, the chimeric protein(s) contain one or more “action” or“effector” domains which are heterologous with respect to the FKBPand/or FRB domains. Effector domains may be selected from a wide varietyof protein domains including DNA binding domains, transcriptionactivation domains, cellular localization domains and signaling domains(i.e., domains which are capable upon clustering or multimerization, oftriggering cell growth, proliferation, differentiation, apoptosis, genetranscription, etc.). A variety of illustrative effector domains whichmay be used in practising this invention are disclosed in the varioussscientific and patent documents cited herein.

[0033] For example, in certain embodiments, one fusion protein containsat least one DNA binding domain (e.g., a GAL4 or ZFHD1 DNA-bindingdomain) and another fusion protein contains at least one transcriptionactivation domain (e.g., a VP16 or p65 transcription activation domain).Ligand-mediated association of the fusion proteins represents theformation of a transcription factor complex and leads to initiation oftranscription of a target gene linked to a DNA sequence recognized by(i.e., capable of binding with) the DNA-binding domain on one of thefusion proteins.

[0034] In other embodiments, one fusion protein contains at least onedomain capable of directing the fusion protein to a particular cellularlocation such as the cell membrane, nucleus, ER or other organelle orcellular component. Localization domains which target the cell membrane,for example, include domains such as a myristoylation site or atransmembrane region of a receptor protein or other membrane-spanningprotein. Another fusion protein can contain a signaling domain capable,upon membrane localization and/or clustering, of activating a cellularsignal transduction pathway. Examples of signaling domains include anintracellular domain of a growth factor or cytokine receptor, anapoptosis triggering domain such as the intracellular domain of FAS orTNF-R1, and domains derived from other intracellular signaling proteinssuch as SOS, Raf, Ick, ZAP-70, etc. A number of signaling proteins aredisclosed in PCT/US94/01617 (see e.g. pages 23-26). In still otherembodiments, each of the fusion proteins contains at least one FRBdomain and at least one FKBP domain, as well as one or more heterologousdomains. Such fusion proteins are capable of homodimerization andtriggering signaling in the presence of the rapalog. In general, domainscontaining peptide sequence endogenous to the host cell are preferred inapplications involving whole organisms. Thus, for human gene therapyapplications, domains of human origin are of particular interest.

[0035] Recombinant nucleic acid constructs encoding the fusion proteinsare also provided, as are nucleic acid constructs capable of directingtheir expression, and vectors containing such constructs for introducingthem into cells, particularly eukaryotic cells, of which yeast andanimal cells are of particular interest. In view of the constituentcomponents of the fusion proteins, the recombinant DNA molecules whichencode them are capable of selectively hybridizing (a) to a DNA moleculeencoding a polypeptide comprising an FRB domain or FKBP domain and (b)to a DNA molecule encoding the heterologous domain or a protein fromwhich the heterologous protein domain was derived. DNAs are alsoencompassed which would be capable of so hybridizing but for thedegeneracy of the genetic code.

[0036] Using nucleic acid sequences encoding the fusion proteins,nucleic acid constructs for directing their expression in eukaryoticcells, and vectors or other means for introducing such constructs intocells, especially animal cells, one may genetically engineer cells,particularly animal cells, preferably mammlian cells, and mostpreferably human cells, for a number of important uses. To do so, onefirst provides an expression vector or nucleic acid construct fordirecting the expression in a eukaryotic (preferably animal) cell of thedesired chimeric protein(s) and then introduces the recombinant DNA intothe cells in a manner permitting DNA uptake and expression of theintroduced DNA in at least a portion of the cells. One may use any ofthe various methods and materials for introducing DNA into cells forheterologous gene expression, a variety of which are well known and/orcommercially available.

[0037] One object of this invention is thus a method for multimerizingfusion proteins, such as described herein, in cells, preferably animalcells. To recap, one of the fusion proteins is capable of binding to theimproved rapalog of this invention and contains at least one FKBP domainand at least one domain heterologous thereto. The second fusion proteincontains at least one FRB domain and at least one domain heterologousthereto and is capable of forming a tripartite complex with the firstfusion protein and one or more molecules of the improved rapalog. Insome embodiments one or more of the heterologous domains present on oneof the fusion proteins are also present on the other fusion protein,i.e., the two fusion proteins have one or more common heterologousdomains. In other embodiments, each fusion protein contains one or moredifferent heterologous domains.

[0038] The method comprises contacting appropriately engineered cellswith the improved rapalog by adding the rapalog to the culture medium inwhich the cells are located or administering the rapalog to the organismin which the cells are located. The cells are preferably eukaryoticcells, more preferably animal cells, and most preferably mammaliancells. Primate cells, especially human cells, are of particularinterest. Administration of the improved rapalog to a human or non-humananimal may be effected using any pharmaceutically acceptable formulationand route of administration. Oral administration of a pharmaceuticallyacceptable composition containing the improved rapalog together with oneor more pharmaceuticaly acceptable carriers, buffers or other excipientsis currently of greatest interest.

[0039] A specific object of this invention is a method, as otherwisedescribed above, for inducing transcription of a target gene in arapalog-dependent manner. The cells typically contain, in addition torecombinant DNAs encoding the two fusion proteins, a target geneconstruct which comprises a target gene operably linked to a DNAsequence which is responsive to the presence of a complex of the fusionproteins with rapamycin or a rapalog. The target gene construct may berecombinant, and the target gene and/or a regulatory nucleic acidsequence linked thereto may be heterologous with respect to the hostcell. In certain embodiments the cells are responsive to contact with animproved rapalog which binds to the FKBP fusion protein and participatesin a complex with a FRB fusion protein with a detectable preference overbinding to endogenous FKBP and/or FRB-containing proteins of the hostcell.

[0040] Another specific object of this invention is a method, asotherwise described above, for inducing cell death in arapalog-dependent manner. In such cells, at least one of theheterologous domains on at least one fusion protein, and usually twofusion proteins, is a domain such as the intracellular domain of FAS orTNF-R1, which, upon clustering, triggers apoptosis of the cell.

[0041] Another specific object of this invention is a method, asotherwise described above, for inducing cell growth, differentiation orproliferation in a rapalog-dependent manner. In such cells, at least oneof the heterologous domains of at least one of the fusion proteins is asignaling domain such as, for example, the intracellular domain of areceptor for a hormone which mediates cell growth, differentiation orproliferation, or a downstream mediator of such a receptor. Cell growth,differentiation and/or proliferation follows clustering of suchsignalling domains. Such clustering occurs in nature following hormonebinding, and in engineered cells of this invention following contactwith an improved rapalog.

[0042] Cells of human origin are preferred for human gene therapyapplications, although cell types of various origins (human or otherspecies) may be used, and may, if desired, be encapsulated within abiocompatible material for use in human subjects.

[0043] Also provided are materials and methods for producing theforegoing engineered cells. This object is met by providing recombinantnucleic acids, typically DNA molecules, encoding the fusion proteins,together with any desired ancillary recombinant nucleic acids such as atarget gene construct, and introducing the recombinant nucleic acidsinto the host cells under conditions permitting nucleic acid uptake bycells. Such transfection may be effected ex vivo, using host cellsmaintained in culture. Cells that are engineered in culture maysubsequently be introduced into a host organism, e.g. in ex vivo genetherapy applications. Doing so thus constitutes a method for providing ahost organism, preferably a human or non-human mammal, which isresponsive (as described herein) to the presence of an improved rapalogas provided herein. Alternatively transfection may be effected in vivo,using host cells present in a human or non-human host organism. In suchcases, the nucleic acid molecules are introduced directly into the hostorganism under conditions permitting uptake of nucleic acids by one ormore of the host organism's cells. This approach thus constitutes analternative method for providing a host organism, preferably a human ornon-human mammal, which is responsive (as described herein) to thepresence of an improved rapalog. Various materials and methods for theintroduction of DNA and RNA into cells in culture or in whole organismsare known in the art and may be adapted for use in practicing thisinvention.

[0044] Other objects are achieved using the engineered cells describedherein. For instance, a method is provided for multimerizing fusionproteins of this invention by contacting cells engineered as describedherein with an effective amount of the improved rapalog permitting therapalog to form a complex with the fusion proteins. In embodiments inwhich multimerization of the fusion proteins triggers transcription of atarget gene, this constitutes a method for activating the expression ofthe target gene. In embodiments in which the fusion proteins contain oneor more signaling domains, this constitutes a method for activating acellular signal transduction pathway. In specific embodiments in whichthe signaling domains are selected based on their ability followingclustering to trigger cell growth, proliferation, diffeentiation or celldeath, improved rapalog-mediated clustering constitutes a method foractuating cell growth, proliferation, diffeentiation or cell death, asthe case may be. These methods may be carried out in cell culture or inwhole organisms, including human patients. In the former case, therapamycin or rapalog is added to the culture medium. In the latter case,the rapamycin or rapalog (which may be in the form of a pharmaceuticalor veterinary composition) is administered to the whole organism, e.g.,orally, parenterally, etc. Preferably, the dose of the improved rapalogadministered to an animal is below the dosage level that would causeundue immunosuppression in the recipient.

[0045] Also disclosed are kits for use in the genetic engineering ofcells or human or non-human animals as described herein. One such kitcontains one or more recombinant nucleic acid constructs encoding fusionproteins of this invention. The recombinant nucleic acid constructs willgenerally be in the form of eukaryotic expression vectors suitable forintroduction into animal cells and capable of directing the expressionof the fusion proteins therein. Such vectors may be viral vectors asdescribed elsewhere herein. The kit may also contain a sample of animproved rapalog of this invention capable of forming a complex with theencoded fusion proteins. The kit may further contain a multimerizationantagonist such as FK506 or some other compound capable of binding toone of the fusion proteins but incapable of forming a complex with both.In certain embodiments, the recombinant nucleic acid constructs encodingthe fusion proteins will contain a cloning site in place of DNA encodingone or more of the heterologous domains, thus permitting thepractitioner to introduce DNA encoding a heterologous domain of choice.In some embodiments the kit may also contain a target gene constructcontaining a target gene or cloning site linked to a DNA sequenceresponsive to the presence of the complexed fusion proteins, asdescribed in more detail elsewhere. The kit may contain a package insertidentifying the enclosed nucleic acid construct(s), and/or instructionsfor introducing the construct(s) into host cells or organisms.

BRIEF DESCRIPTION OF THE FIGURES

[0046]FIG. 1 demonstrates the ability of 13-F-rapalogs (compounds 79 and108, synthesized as described in Examples 6.1 and 6.21, respectively) tostimulate expression of a DNA sequence encoding secreted alkalinephosphatase (“SEAP”) in HT1080 cells engineered as described in Example7.

[0047]FIG. 2 depicts the results of transcription assays using rapalogs42, 53, 69 and 96, synthesized as described herein, as dimerizer.Rapalog s were tested in cells expressing wild-type FRB (FIGS. 2A and2C) as well as in cells expressing a mutant FRB in which Thr 2098 wasreplaced by Leu (FIGS. 2B and 2D) or by Phe (FIG. 2E).

DETAILED DESCRIPTION OF THE INVENTION

[0048] Definitions

[0049] The definitions and orienting information below will be helpfulfor a full understanding of this document.

[0050] FRB domains are polypeptide regions (protein “domains”),typically of at least about 89 amino acid residues, which are capable offorming a tripartite complex with an FKBP protein and rapamycin (or animproved rapalog of this invention). FRB domains are present in a numberof naturally occurring proteins, including FRAP proteins (also referredto in the literature as “RAPT1” or “RAFT”) from human and other species;yeast proteins including Tor1 and Tor2; and a Candida FRAP homolog.Information concerning the nucleotide sequences, cloning, and otheraspects of these proteins is already known in the art, permitting thesynthesis or cloning of DNA encoding the desired FRB peptide sequence,e.g., using well known methods and PCR primers based on publishedsequences. protein source reference/sequence accession numbers humanFRAP Brown et al, 1994, Nature 369, 756-758; GenBank accession # L34075,NCBI Seq ID 508481; Chiu et al, 1994, PNAS USA 91, 12574-12578; Chen etal, 1995, PNAS USA 92, 4947-4951 murine RAPT1 Chiu et al, supra. yeastTor1 Helliwell et al, 1994, Mol Cell Biol 5, 105-118; EMBL Accession#X74857, NCBI Seq Id #468738 yeast Tor 2 Kunz et al, 1993, Cell 73,585-596; EMBL Accession #X71416, NCBI Seq ID 298027 Candida TORWO95/33052 (Berlin et al)

[0051] FRB domains for use in this invention generally contain at leastabout 89-100 amino acid residues. FIG. 2 of Chiu et al, supra, displaysa 160-amino acid span of human FRAP, murine FRAP, S. cerevisiae TOR1 andS. cerevisiae TOR2 encompassing the conserved FRB region. Typically theFRB sequence selected for use in fusion proteins of this invention willspan at least the 89-amino acid sequence Glu-39 through Lys/Arg-127, asthe sequence is numbered in that figure. For reference, using thenumbering of Chen et al or Sabitini et al, the 89-amino acid sequence isnumbered Glu-2025 through Lys-2113 in the case of human FRAP, Glu-1965through Lys-2053 in the case of Tor2, and Glu-1962 through Arg-2050 inthe case of Tor1. An FRB domain for use in fusion proteins of thisinvention will be capable of binding to a complex of an FKBP proteinbound to rapamycin or an improved rapalog of this invention (as may bedetermined by any means, direct or indirect, for detecting such binding,including, for example, means for detecting such binding employed in theFRAP/RAFT/RAPT and Tor-related references cited herein). The peptidesequence of such an FRB domain comprises (a) a naturally occurringpeptide sequence spanning at least the indicated 89-amino acid region ofthe proteins noted above or corresponding regions of homologousproteins; (b) a variant of a naturally occurring FRB sequence in whichup to about ten (preferably 1-5, more preferably 1-3, and in someembodiments just one) amino acids of the naturally-occurring peptidesequence have been deleted, inserted, or replaced with substitute aminoacids; or (c) a peptide sequence encoded by a DNA sequence capable ofselectively hybridizing to a DNA molecule encoding a naturally occurringFRB domain or by a DNA sequence which would be capable, but for thedegeneracy of the genetic code, of selectively hybridizing to a DNAmolecule encoding a naturally occurring FRB domain.

[0052] FKBPs (FK506 binding proteins) are the cytosolic receptors formacrolides such as FK506, FK520 and rapamycin and are highly conservedacross species lines. For the purpose of this disclosure, FKBPs areproteins or protein domains which are capable of binding to rapamycin orto an improved rapalog of this invention and further forming atripartite complex with an FRB-containing protein. An FKBP domain mayalso be referred to as a “rapamycin binding domain”. Informationconcerning the nucleotide sequences, cloning, and other aspects ofvarious FKBP species is already known in the art, permitting thesynthesis or cloning of DNA encoding the desired FKBP peptide sequence,e.g., using well known methods and PCR primers based on publishedsequences. See e.g. Staendart et al, 1990, Nature 346, 671-674 (humanFKBP12); Kay, 1996, Biochem. J. 314, 361-385 (review). Homologous FKBPproteins in other mammalian species, in yeast, and in other organsimsare also known in the art and may be used in the fusion proteinsdisclosed herein. See e.g. Kay, 1996, Biochem. J. 314, 361-385 (review).The size of FKBP domains for use in this invention varies, depending onwhich FKBP protein is employed. An FKBP domain of a fusion protein ofthis invention will be capable of binding to rapamycin or an improvedrapalog of this invention and participating in a tripartite complex withan FRB-containing protein (as may be determined by any means, direct orindirect, for detecting such binding). The peptide sequence of an FKBPdomain of an FKBP fusion protein of this invention comprises (a) anaturally occurring FKBP peptide sequence, preferably derived from thehuman FKBP12 protein (exemplified below) or a peptide sequence derivedfrom another human FKBP, from a murine or other mammalian FKBP, or fromsome other animal, yeast or fungal FKBP; (b) a variant of a naturallyoccurring FKBP sequence in which up to about ten (preferably 1-5, morepreferably 1-3, and in some embodiments just one) amino acids of thenaturally-occurring peptide sequence have been deleted, inserted, orreplaced with substitute amino acids; or (c) a peptide sequence encodedby a DNA sequence capable of selectively hybridizing to a DNA moleculeencoding a naturally occurring FKBP or by a DNA sequence which would becapable, but for the degeneracy of the genetic code, of selectivelyhybridizing to a DNA molecule encoding a naturally occurring FKBP.

[0053] “Capable of selectively hybridizing” as that phrase is usedherein means that two DNA molecules are susceptible to hybridizationwith one another, despite the presence of other DNA molecules, underhybridization conditions which can be chosen or readily determinedempirically by the practitioner of ordinary skill in this art. Suchtreatments include conditions of high stringency such as washingextensively with buffers containing 0.2 to 6×SSC, and/or containing 0.1%to 1% SDS, at temperatures ranging from room temperature to 65-75° C.See for example F. M. Ausubel et al., Eds, Short Protocols in MolecularBiology, Units 6.3 and 6.4 (John Wiley and Sons, New York, 3d Edition,1995).

[0054] The terms “protein”, “polypeptide” and “peptide” are usedinterchangeably herein.

[0055] “Nucleic acid constructs”, as that term is used herein, denotenucleic acids (usually DNA, but also encompassing RNA, e.g. in aretroviral delivery system) used in the practice of this invention whichare generally recombinant, as that term is defined below, and which mayexist in free form (i.e., not covalently linked to other nucleic acidsequence) or may be present within a larger molecule such as a DNAvector, retroviral or other viral vector or a chromosome of agenetically engineered host cell. Nucleic acid constructs of particularinterest are those which encode fusion proteins of this invention orwhich comprise a target gene and expression control elements. Theconstruct may further include nucleic acid portions comprising one ormore of the following elements relevant to regulation of transcription,translation, and/or other processing of the coding region or geneproduct thereof: transcriptional promoter and/or enhancer sequences, aribosome binding site, introns, etc.

[0056] “Recombinant”, “chimeric” and “fusion”, as those terms are usedherein, denote materials comprising various component domains, sequencesor other components which are mutually heterologous in the sense thatthey do not occur together in the same arrangement, in nature. Morespecifically, the component portions are not found in the samecontinuous polypeptide or nucleotide sequence or molecule in nature, atleast not in the same cells or order or orientation or with the samespacing present in the chimeric protein or recombinant DNA molecule ofthis invention.

[0057] “Transcription control element” denotes a regulatory DNAsequence, such as initiation signals, enhancers, and promoters, whichinduce or control transcription of protein coding sequences with whichthey are operably linked. The term “enhancer” is intended to includeregulatory elements capable of increasing, stimulating, or enhancingtranscription from a promoter. Such transcription regulatory componentscan be present upstream of a coding region, or in certain cases (e.g.enhancers), in other locations as well, such as in introns, exons,coding regions, and 3′ flanking sequences.

[0058] “Dimerization”, “oligomerization” and “multimerization” are usedinterchangeably herein and refer to the association or clustering of twoor more protein molecules, mediated by the binding of a drug to at leastone of the proteins. In preferred embodiments, the multimerization ismediated by the binding of two or more such protein molecules to acommon divalent or multivalent drug. The formation of a complexcomprising two or more protein molecules, each of which containing oneor more FKBP domains, together with one or more molecules of an FKBPligand which is at least divalent (e.g. FK1012 or AP1510) is an exampleof such association or clustering. In cases where at least one of theproteins contains more than one drug binding domain, e.g., where atleast one of the proteins contains three FKBP domains, the presence of adivalent drug leads to the clustering of more than two proteinmolecules. Embodiments in which the drug is more than divalent (e.g.trivalent) in its ability to bind to proteins bearing drug bindingdomains also can result in clustering of more than two proteinmolecules. The formation of a tripartite complex comprising a proteincontaining at least one FRB domain, a protein containing at least oneFKBP domain and a molecule of rapamycin is another example of suchprotein clustering. In certain embodiments of this invention, fusionproteins contain multiple FRB and/or FKBP domains. Complexes of suchproteins may contain more than one molecule of rapamycin or a derivativethereof or other dimerizing agent and more than one copy of one or moreof the constituent proteins. Again, such multimeric complexes are stillreferred to herein as tripartite complexes to indicate the presence ofthe three types of constituent molecules, even if one or more arerepresented by multiple copies. The formation of complexes containing atleast one divalent drug and at least two protein molecules, each ofwhich contains at least one drug binding domain, may be referred to as“oligomerization” or “multimerization”, or simply as “dimerization”,“clustering” or “association”.

[0059] “Dimerizer” denotes an improved rapalog of this invention whichbrings together two or more proteins in a multimeric complex.

[0060] “Activate” as applied herein to the expression or transcriptionof a gene denotes a directly or indirectly observable increase in theproduction of a gene product.

[0061] “Genetically engineered cells” denotes cells which have beenmodified (“transduced”) by the introduction of recombinant orheterologous nucleic acids (e.g. one or more DNA constructs or their RNAcounterparts) and further includes the progeny of such cells whichretain part or all of such genetic modification.

[0062] A “therapeutically effective dose” of an improved rapalog of thisinvention denotes a treatment- or prophylaxis-effective dose, e.g., adose which yields detectable target gene transcription or cell growth,proliferation, differentiation, death, etc. in the geneticallyengineered cell, or a dose which is predicted to be treatment- orprophylaxis-effective by extrapolation from data obtained in animal orcell culture models. A therapeutically effective dose is ususallypreferred for the treatment of a human or non-human mammal.

[0063] This invention involves methods and materials for multimerizingchimeric proteins in genetically engineered cells using improvedrapalogs. The design and implementation of various dimerization-basedbiological switches has been reported, inter alia, in Spencer et al andin various international patent applications cited herein. Otheraccounts of successful application of this general approach have alsobeen reported. Chimeric proteins containing an FRB domain fused to aneffector domain has also been disclosed in Rivera et al, 1996, NatureMedicine 2, 1028-1032 and in WO 96/41865 (Clackson et al) and WO95/33052 (Berlin et al). As noted previously, the fusion proteins aredesigned such that association of the effector domains, throughligand-mediated “dimerization” or “multimerization” of the fusionproteins which contain them, triggers a desired biological event such astranscription of a desired gene, cell death, cell proliferation, etc.For example, clustering of chimeric proteins containing an action domainderived from the intracellular portion of the T cell receptor CD3 zetadomain triggers transcription of a gene under the transcriptionalcontrol of the IL-2 promoter or promoter elements derived therefrom. Inother embodiments, the action domain comprises a domain derived from theintracellular portion of a protein such as FAS or the TNF-alpha receptor(TNFalpha-R1), which are capable, upon oligomerization, of triggeringapoptosis of the cell. In still other embodiments, the action domainscomprise a DNA-binding domain such as GAL4 or ZFHD1 and a transcriptionactivation domain such as VP16 or p65, paired such that oligomerizationof the chimeric proteins represents assembly of a transcription factorcomplex which triggers transcription of a gene linked to a DNA sequencerecognized by (capable of specific binding interaction with) the DNAbinding domain.

[0064] Chimeric proteins containing one or more ligand-binding domainsand one or more action domains, e.g. for activation of transcription ofa target gene, triggering cell death or other signal transductionpathway, cellular localization, etc., are disclosed in PCT/US94/01617,PCT/US94/08008 and Spencer et al, supra. The design and use of suchchimeric proteins for ligand-mediated gene-knock out and forligand-mediated blockade of gene expression or inhibition of geneproduct function are disclosed in PCT/US95/10591. Novel DNA bindingdomains and DNA sequences to which they bind which are useful inembodiments involving regulated transcription of a target gene aredisclosed, e.g., in Pomeranz et al, 1995, Science 267:93-96. Thosereferences provide substantial information, guidance and examplesrelating to the design, construction and use of DNA constructs encodinganalogous chimeras, target gene constructs, and other aspects which mayalso be useful to the practitioner of the subject invention.

[0065] By appropriate choice of chimeric proteins, this inventionpermits one to activate the transcription of a desired gene; actuatecell growth, proliferation, differentiaion or apoptosis; or triggerother biological events in engineered cells in a rapalog-dependentmanner analogous to the systems described in the patent documents andother references cited above. The engineered cells, preferably animalcells, may be growing or maintained in culture or may be present withinwhole organisms, as in the case of human gene therapy, transgenicanimals, and other such applications. The rapalog is administered to thecell culture or to the organism containing the engineered cells, as thecase may be, in an amount effective to multimerize the FKBP fusionproteins and FRB fusion proteins (as may be observed indirectly bymonitoring target gene transcription, apoptosis or other biologicalprocess so triggered). In the case of administration to whole organisms,the rapalog may be administered in a composition containing the rapalogand one or more acceptable verterinary or pharmaceutical diluents and/orexcipients.

[0066] A compound which binds to one of the chimeric proteins but doesnot form tripartite complexes with both chimeric proteins may be used asa multimerization antagonist. As such it may be administered to theengineered cells, or to organisms containing them (preferably in acomposition as described above in the case of administration to wholeanimals), in an amount effective for blocking or reversing the effect ofthe rapalog, i.e. for preventing, inhibiting or disruptingmultimerization of the chimeras. For instance, FK506, FK520 or any ofthe many synthetic FKBP ligands which do not form tripartite complexeswith FKBP and FRAP may be used as an antagonist.

[0067] One important aspect of this invention provides materials andmethods for rapalog-dependent, direct activation of transcription of adesired gene. In one such embodiment, a set of two or more differentchimeric proteins, and corresponding DNA constructs capable of directingtheir expression, is provided. One such chimeric protein contains as itsaction domain(s) one or more transcriptional activation domains. Theother chimeric protein contains as its action domain(s) one or moreDNA-binding domains. A rapalog of this invention is capable of bindingto both chimeras to form a dimeric or multimeric complex thus containingat least one DNA binding domain and at least one transcriptionalactivating domain. Formation of such complexes leads to activation oftranscription of a target gene linked to, and under the transcriptionalcontrol of, a DNA sequence to which the DNA-binding domain is capable ofbinding, as can be observed by monitoring directly or indirectly thepresence or concentration of the target gene product.

[0068] Preferably the DNA binding domain, and a chimera containing it,binds to its recognized DNA sequence with sufficient selectivity so thatbinding to the selected DNA sequence can be observed (directly orindirectly) despite the presence of other, often numerous other, DNAsequences. Preferably, binding of the chimera comprising the DNA-bindingdomain to the selected DNA sequence is at least two, more preferablythree and even more preferably more than four orders of magnitudegreater than binding to any one alternative DNA sequence, as measured byin vitro binding studies or by measuring relative rates or levels oftranscription of genes associated with the selected DNA sequence ascompared with any alternative DNA sequences.

[0069] Cells which have been genetically engineered to contain such aset of constructs, together with any desired accessory constructs, maybe used in applications involving ligand-mediated, regulated actuationof the desired biological event, be it regulated transcription of adesired gene, regulated triggering of a signal transduction pathway suchas the triggering of apoptosis, or another event. Cells engineered forregulatable expression of a target gene, for instance, can be used forregulated production of a desired protein (or other gene product)encoded by the target gene. Such cells may be grown in culture byconventional means. Addition of the rapalog to the culture mediumcontaining the cells leads to expression of the target gene by the cellsand production of the protein encoded by that gene. Expression of thegene and production of the protein can be turned off by withholdingfurther multimerization agent from the media, by removing residualmultimerization agent from the media, or by adding to the medium amultimerization antagonist reagent.

[0070] Engineered cells of this invention can also be produced and/orused in vivo, to modify whole organisms, preferably animals, especiallyhumans, e.g. such that the cells produce a desired protein or otherresult within the animal containing them. Such uses include gene therapyapplications.

[0071] Embodiments involving regulatable actuation of apoptosis provideengineered cells susceptible to rapalog-inducible cell death. Suchengineered cells can be eliminated from a cell culture or host organismafter they have served their intended purposed (e.g. production of adesired protein or other product), if they have or develop unwantedproperties, or if they are no longer useful, safe or desired.Elimination is effected by adding the rapalog to the medium oradministering it to the host organism. In such cases, the action domainsof the chimeras are protein domains such as the intracellular domains ofFAS or TNF-R1, downstream components of their signaling pathways orother protein domains which upon oligomerization trigger apoptosis.

[0072] This invention thus provides materials and methods for achievinga biological effect in cells in response to the addition of a rapalog ofthis invention. The method involves providing cells engineered asdescribed herein and exposing the cells to the rapalog.

[0073] For example, this invention provides a method for activatingtranscription of a target gene in cells. The method involves providingcells containing (a) DNA constructs encoding a set of chimeric proteinsof this invention capable upon rapalog-mediated multimerization ofinitiating transcription of a target gene and (b) a target gene linkedto an associated cognate DNA sequence responsive to the multimerizationevent (e.g. a DNA sequence recognized, i.e., capable of binding with, aDNA-binding domain of a foregoing chimeric protein. The method involvesexposing the cells to a rapalog capable of binding to the chimericproteins in an amount effective to result in expression of the targetgene. In cases in which the cells are growing in culture, exposing thecells to the rapalog may be effected by adding the rapalog to theculture medium. In cases in which the cells are present within a hostorganism, exposing them to the rapalog is effected by administering therapalog to the host organism. For instance, in cases in which the hostorganism is a human or non-human, the rapalog may be administered to thehost organism by oral, bucal, sublingual, transdermal, subcutaneous,intramuscular, intravenous, intra-joint or inhalation administration inan appropriate vehicle therefor. Again, depending on the design of theconstructs for the chimeric proteins and of any accessory constructs,the rapalog-mediated biological event may be activation of a cellularfunction such as signal transduction leading to cell growth, cellproliferation, gene transcription, or apoptosis; deletion of a gene ofinterest, blockade of expression of a gene of interest, or inhibition offunction of a gene product of interest; direct transcription of a geneof interest; etc.

[0074] This invention further encompasses a pharmaceutical compositioncomprising a rapalog of this invention in admixture with apharmaceutically acceptable carrier and optionally with one or morepharmaceutically acceptable excipients. Such pharmaceutical compositionscan be used to promote multimerization of chimeras of this invention inengineered cells in whole animals, e.g. in human gene therapyapplications to achieve any of the objectives disclosed herein.

[0075] Said differently, this invention provides a method for achievingany of those objectives, e.g. activation of transcription of a targetgene (typically a heterologous gene for a therapeutic protein), cellgrowth or proliferation, cell death or some other selected biologicalevent, in an animal, preferably a human patient, in need thereof andcontaining engineered cells of this invention. That method involvesadministering to the animal a pharmaceutical composition containing therapalog by a route of administration and in an amount effective to causemultimerization of the chimeric proteins in at least a portion of theengineered cells. Multimerization may be detected indirectly bydetecting the occurrence of target gene expression; cell growth,proliferation or death; or other objective for which the chimeras weredesigned and the cells genetically engineered.

[0076] This invention further encompasses a pharmaceutical compositioncomprising a multimerization antagonist of this invention in admixturewith a pharmaceutically acceptable carrier and optionally with one ormore pharmaceutically acceptable excipients for inhibiting or otherwisereducing, in whole or part, the extent of multimerization of chimericproteins in engineered cells of this invention in a subject, and thusfor de-activating the transcription of a target gene, for example, orturning off another biological result of this invention. Thus, the useof the multimerizing rapalogs and of the multimerization antagonistreagents to prepare pharmaceutical compositions and achieve theirpharmacologic results is encompassed by this invention.

[0077] Also disclosed is a method for providing a host organism,preferably an animal, typically a non-human mammal or a human subject,responsive to a rapalog of this invention. The method involvesintroducing into the organism cells which have been engineered inaccordance with this invention, i.e. containing one or more nucleic acidconstructs encoding the chimeric proteins, and so forth. The engineeredcells may be encapsulated using any of a variety of materials andmethods before being introduced into the host organism. Alternatively,one can introduce the nucleic acid constructs of this invention into ahost organism, e.g. a mammal, under conditions permitting incorporationthereof into one or more cells of the host mammal, e.g. using viralvectors, introduction of DNA by injection or via catheter, etc.

[0078] Also provided are kits for producing cells responsive to arapalog of this invention. One such kit contains one or more nucleicacid constructs encoding and capable of directing the expression ofchimeras which, upon rapalog-mediated oligomerization, trigger thedesired biological response. The kit may contain a quantity of a rapalogcapable of multimerizing the chimeric protein molecules encoded by theconstruct(s) of the kit, and may contain in addition a quantity of amultimerization antagonist. The kit may further contain a nucleic acidconstruct encoding a target gene (or cloning site) linked to a cognateDNA sequence which is recognized by the dimerized chimeric proteinspermitting transcription of a gene linked to that cognate DNA sequencein the presence of multimerized chimeric protein molecules. Theconstructs may be associated with one or more selection markers forconvenient selection of transfectants, as well as other conventionalvector elements useful for replication in prokaryotes, for expression ineukaryotes, and the like. The selection markers may be the same ordifferent for each different construct, permitting the selection ofcells which contain each such construct(s).

[0079] The accessory construct for introducing into cells a target genein association with a cognate DNA sequence may contain a cloning site inplace of a target gene. A kit containing such a construct permits theengineering of cells for regulatable expression of a gene to be providedby the practitioner.

[0080] Other kits of this invention may contain one or two (or more)nucleic acid constructs for chimeric proteins in which one or morecontain a cloning site in place of the transcriptional activator or DNAbinding protein, permitting the user to insert whichever such domains/he wishes. Such a kit may optionally include other elements asdescribed above, e.g. a nucleic construct for a target gene with orwithout a cognate DNA sequence for a pre-selected DNA binding domain.

[0081] Any of the kits may also contain positive control cells whichwere stably transformed with constructs of this invention such that theyexpress a reporter gene (for CAT, beta-galactosidase or any convenientlydetectable gene product) in response to exposure of the cells to therapalog. Reagents for detecting and/or quantifying the expression of thereporter gene may also be provided.

[0082] For further information and guidance on the design, constructionand use of such systems or components thereof which may be adapted foruse in practising the subject invention, reference to the followingpublications is suggested: Spencer et al, 1993, supra; Rivera et al,1996, supra; Spencer et al, 1996, Current Biology 6, 839-847; Luo et al,1996, Nature, 383, 181-185; Ho et al, 1996, Nature 382, 822-826; Belshawet al, 1996, Proc. Natl. Acad. Sci. USA 93, 4604-4607; Spencer, 1996,TIG 12(5), 181-187; Spencer et al, 1995, Proc., Natl. Acad. Sci. USA 92,9805-9809; Holsinger et al, 1995, Proc. Natl. Acad. Sci. USA 92,9810-9814; Pruschy et al, 1994, Chemistry & Biology 1(3),163-172; andpublished international patent applications WO 94/18317, WO 95/02684, WO95/33052, WO 96/20951 and WO 96/41865.

[0083] A key focus of the subject invention is the use of improvedrapalogs as mediators of protein-protein interactions in applicationsusing FKBP and FRB fusion proteins such as described above and elsewhereherein. The improved rapalogs may be used in the various applications ofthe underlying dimerization-based technology, including triggeringbiological events in genetically engineered cells grown or maintained inculture or present in whole organisms, including humans and othermammals. The improved rapalogs may thus be useful as research reagentsin biological experiments in vitro, in experiments conducted on animalscontaining the genetically engineered cells, and as prophylactic ortherapeutic agents in animal and human health care in subjectscontaining genetically engineered cells.

[0084] Rapalogs

[0085] “Rapalogs” as that term is used herein denotes a class ofcompounds comprising the various analogs, homologs and derivatives ofrapamycin and other compounds related structurally to rapamycin.“Rapalogs” include compounds other than rapamycin which comprise thesubstructure shown in Formula I, bearing any number of a variety ofsubstituents, and optionally unsaturated at one or more carbon-carbonbonds unless specified to the contrary herein.

[0086] Rapalogs include, among others, variants of rapamycin having oneor more of the following modifications relative to rapamycin:demethylation, elimination or replacement of the methoxy at C7, C42and/or C29; elimination, derivatization or replacement of the hydroxy atC13, C43 and/or C28; reduction, elimination or derivatization of theketone at C14, C24 and/or C30; replacement of the 6-membered pipecolatering with a 5-membered prolyl ring; and elimination, derivatization orreplacement of one or more substituents of the cyclohexyl ring orreplacement of the cyclohexyl ring with a substituted or unsubstitutedcyclopentyl ring. Rapalogs, as that term is used herein, do not includerapamycin itself, and preferably do not contain an oxygen bridge betweenC1 and C30. Illustrative examples of rapalogs are disclosed in thedocuments listed in Table I. Examples of rapalogs modified at C7 areshown in Table II. TABLE I WO9710502 WO9418207 WO9304680 US5527907US5225403 WO9641807 WO9410843 WO9214737 US5484799 US5221625 WO9635423WO9409010 WO9205179 US5457194 US5210030 WO9603430 WO94/04540 US5604234US5457182 US5208241 WO9600282 WO9402485 US5597715 US5362735 US5200411WO9516691 WO9402137 US5583139 US5324644 US5198421 WO9515328 WO9402136US5563172 US5318895 US5147877 WO9507468 WO9325533 US5561228 US5310903US5140018 WO9504738 WO9318043 US5561137 US5310901 US5116756 WO9504060WO9313663 US5541193 US5258389 US5109112 WO9425022 WO9311130 US5541189US5252732 US5093338 WO9421644 WO9310122 US5534632 US5247076 US5091389

[0087] TABLE II Illustrative C7 rapalog structures

[0088] Other illustrative rapalogs include those depicted in Table III:TABLE III

[0089] Rapalogs of particular interest for the practice of variousaspects of this invention include compounds of formula II:

[0090] one of R^(C7a) and R^(C7b) is H and the other is —H, halo, —R²,—OR¹, —SR¹, —OC(O)R¹, —OC(O)NHR¹, —NHR¹, —NR¹R², —NHC(O)R¹, or—NH—SO₂—R¹ where R²=aliphatic, heteroaliphatic, aryl, heteroaryl oralkylaryl (e.g. benzyl or substituted benzyl),

[0091] R^(C30) is halo, —OR³ or (═O),

[0092] R^(C24) is ═O, ═NR⁴═NOR⁴, ═NNHR⁴, —NHOR⁴, —NHNHR⁴, —OR⁴, —OC(O)R⁴or —OC(O)NR⁴, halo or —H,

[0093] R^(C13) and R^(C28) are independently H, halo, —OR³, —OR⁵,—OC(O)R⁵, —OC(O)NHR⁵, —SR⁵, —SC(O)R⁵, —SC(O)NHR⁵, —NR⁵R^(5′) or—N(R⁵)(CO)R^(5′)

[0094] R^(C14) is ═O, —OR⁶, —NR⁶, —H, —NC(O)R⁶, —OC(O)R⁶ or —OC(O)NR⁶

[0095] R³ is H, —R⁷, —C(O)R⁷ or —C(O)NHR⁷ or a cyclic moiety (e.g.,carbonate) bridging C28 and C30

[0096] R^(C29) is H or OR¹¹ (e.g., OH or OMe)

[0097] where each substituent may be present in either stereochemicalorientation unless otherwise indicated, and where each occurrence of R¹,R⁴, R⁵, R⁶, R⁷, R⁹, R¹⁰ and R¹¹ is independently selected from H,aliphatic, heteroaliphatic, aryl and heteroaryl; and R⁸ is H, halo, —CN,═O, —OH, —NR⁹R¹⁰, OSO₂CF₃, OSO₂F, OSO₂R⁴, OCOR⁴, OCONR^(4′)R⁵, orOCON(OR^(4′))R^(5′).

[0098] Some rapalogs of Formula II differ from rapamycin only in thatR^(C13) is —OMe and R^(C14) is H; R^(C14) is —OH, —O(CO)NHMe, —O—CH₂—(i.e., a spiro epoxide), ═NCH₂CH₂—OH, or —O-phenyl; R^(C13) is—NHC(O)Me; R⁴ is Me; R^(C24) is ═NR or —NHR, where R is —OH, O-alkyl(methyl, ethyl, isobutyl, benzyl), —NH-alkyl or an O-carboxymethyloximeor O-carboxamidomethyloxime at C24; R^(C24) is —O(CO)NHCH(CH₃)₂; R^(C24)is —O(CO)NC(O)CH₂CH₂C(O) and R^(C28) O-TBDMS; R^(C28) or R^(C30) is—OC(O)R, or R^(C28) and R^(C30) together comprise —OC(O)O— linking C28and C30 in a six-membered ring; or R^(C7a) or R^(C7b) is isopropoxyl,—S-phenyl, 2-thiophen-yl, 3-indol-yl or allyl or methallyl. See TableIII and Liberles et al, 1997, Proc Natl Acad Sci USA 94:7825-7830.Rapalogs other than the foregoing, i.e., which contain alternativemodifications or combinations of modifications relative to the structureof rapamycin, are preferred for use in practising the subject invention.Thus the improved rapalogs of this invention are rapalogs other thanthose depicted in Table III.

[0099] In rapamycin, R^(C7a) is —OMe; R^(C7b) is H; R^(C14), R^(C24) andR^(C30) are each (═O); R^(C13) and R^(C28) are each —OH; R^(C29) is OMe;and R³ and R⁴ are each H, all with the stereoisomerism as shown onpage 1. Rapalogs useful in practicing this invention may containsubstituents in any of the possible stereoisomeric orientations, and maycomprise one stereoisomer substantially free of other stereoisomers(>90%, and preferably >95%, free from other stereoisomers on a molarbasis) or may comprise a mixture of stereoisomers.

[0100] Also included are pharmaceutically acceptable derivatives of theforegoing compounds, where the phrase “pharmaceutically acceptablederivative” denotes any pharmaceutically acceptable salt, ester, or saltof such ester, of such compound, or any other adduct or derivativewhich, upon administration to a patient, is capable of providing(directly or indirectly) a rapalog as described herein, or a metaboliteor residue thereof. Pharmaceutically acceptable derivatives thus includeamong others pro-drugs of the rapalogs. A pro-drug is a derivative of acompound, usually with significantly reduced pharmacological activity,which contains an additional moiety which is susceptible to removal invivo yielding the parent molecule as the pharmacologically activespecies. An example of a pro-drug is an ester which is cleaved in vivoto yield a compound of interest. Various pro-drugs of rapamycin and ofother compounds, and materials and methods for derivatizing the parentcompounds to create the pro-drugs, are known and may be adapted to thepresent invention.

[0101] The term “aliphatic” as used herein includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, cyclic, orpolycyclic aliphatic hydrocarbons, which are optionally substituted withone or more functional groups. Unless otherwise specified, alkyl, otheraliphatic, alkoxy and acyl groups preferably contain 1-8, and in manycases 1-6, contiguous aliphatic carbon atoms. Illustrative aliphaticgroups thus include, for example, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, —CH₂-cyclopropyl, allyl, n-butyl, sec-butyl, isobutyl,tert-butyl, cyclobutyl, —CH₂-cyclobutyl, n-pentyl, sec-pentyl,isopentyl, tert-pentyl, cyclopentyl, —CH₂-cyclopentyl, n-hexyl,sec-hexyl, cyclohexyl, —CH₂-cyclohexyl moieties and the like, whichagain, may bear one or more substituents.

[0102] Examples of substituents include: —OH, —OR², —SH, —SR^(2′), —CHO,═O, —COOH (or ester, carbamate, urea, oxime or carbonate thereof), —NH₂(or substituted amine, amide, urea, carbamate or guanidino derivativetherof), halo, trihaloalkyl, cyano, —SO₂—CF₃, —OSO₂F, —OS(O)₂R¹¹,—SO₂—NHR¹¹, —NHSO₂—R¹¹, sulfate, sulfonate, aryl and heteroarylmoieties. Aryl and heteroaryl substituents may themselves be substitutedor unsubstituted (e.g. mono-, di- and tri-alkoxyphenyl;methylenedioxyphenyl or ethylenedioxyphenyl; halophenyl; or-phenyl-C(Me)₂—CH₂—O—CO—[C3-C6] alkyl or alkylamino).

[0103] The term “aliphatic” is thus intended to include alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.

[0104] As used herein, the term “alkyl” includes both straight, branchedand cyclic alkyl groups. An analogous convention applies to othergeneric terms such as “alkenyl”, “alkynyl” and the like. Furthermore, asused herein, the language “alkyl”, “alkenyl”, “alkynyl” and the likeencompasses both substituted and unsubstituted groups.

[0105] The term “alkyl” refers to groups usually having one to eight,preferably one to six carbon atoms. For example, “alkyl” may refer tomethyl, ethyl, n-propyl, isopropyl, cyclopropyl, butyl, isobutyl,sec-butyl, tert-butyl, cyclobutyl, pentyl, isopentyl tert-pentyl,cyclopentyl, hexyl, isohexyl, cyclohexyl, and the like. Suitablesubstituted alkyls include, but are not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substitutedbenzyl and the like.

[0106] The term “alkenyl” refers to groups usually having two to eight,preferably two to six carbon atoms. For example, “alkenyl” may refer toprop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl,hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. The language“alkynyl,” which also refers to groups having two to eight, preferablytwo to six carbons, includes, but is not limited to, prop-2-ynyl,but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3-methylpent-4-ynyl, hex-2-ynyl,hex-5-ynyl, and the like.

[0107] The term “cycloalkyl” as used herein refers specifically togroups having three to seven, preferably three to ten carbon atoms.Suitable cycloalkyls include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like, which, asin the case of other aliphatic or heteroaliphatic or heterocyclicmoieties, may optionally be substituted.

[0108] The term “heteroaliphatic” as used herein refers to aliphaticmoieties which contain one or more oxygen, sulfur, nitrogen, phosphorousor silicon atoms, e.g., in place of carbon atoms. Heteroaliphaticmoieties may be branched, unbranched or cyclic and include heterocyclessuch as morpholino, pyrrolidinyl, etc.

[0109] The term “heterocycle” as used herein refers to cyclicheteroaliphatic groups and preferably three to ten ring atoms total,includes, but is not limited to, oxetane, tetrahydrofuranyl,tetrahydropyranyl, aziridine, azetidine, pyrrolidine, piperidine,morpholine, piperazine and the like.

[0110] The terms “aryl” and “heteroaryl” as used herein refer to stablemono- or polycyclic, heterocyclic, polycyclic, and polyheterocyclicunsaturated moieties having 3-14 carbon atoms which may be substitutedor unsubstituted. Substituents include any of the previously mentionedsubstituents. Non-limiting examples of useful aryl ring groups includephenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl, trialkoxyphenyl,alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl, phenanthro and thelike. Examples of typical heteroaryl rings include 5-membered monocyclicring groups such as thienyl, pyrrolyl, imidazolyl, pyrazolyl, furyl,isothiazolyl, furazanyl, isoxazolyl, thiazolyl and the like; 6-memberedmonocyclic groups such as pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,triazinyl and the like; and polycyclic heterocyclic ring groups such asbenzo[b]thienyl, naphtho[2,3-b]thienyl, thianthrenyl, isobenzofuranyl,chromenyl, xanthenyl, phenoxathienyl, indolizinyl, isoindolyl, indolyl,indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl, naphthyridinyl,quinoxalinyl, quinazolinyl, benzothiazole, benzimidazole,tetrahydroquinoline cinnolinyl, pteridinyl, carbazolyl, beta-carbolinyl,phenanthridinyl, acridinyl, perimidinyl, phenanthrolinyl, phenazinyl,isothiazolyl, phenothiazinyl, phenoxazinyl, and the like (see e.g.Katritzky, Handbook of Heterocyclic Chemistry). The aryl or heteroarylmoieties may be substituted with one to five members selected from thegroup consisting of hydroxy, C1-C8 alkoxy, C1-C8 branched orstraight-chain alkyl, acyloxy, carbamoyl, amino, N-acylamino, nitro,halo, trihalomethyl, cyano, and carboxyl. Aryl moieties thus include,e.g. phenyl; substituted phenyl bearing one or more substituentsselected from groups including: halo such as chloro or fluoro, hydroxy,C1-C6 alkyl, acyl, acyloxy, C1-C6 alkoxy (such as methoxy or ethoxy,including among others dialkoxyphenyl moieties such as 2,3-, 2,4-, 2,5-,3,4- or 3,5-dimethoxy or diethoxy phenyl or such asmethylenedioxyphenyl, or 3-methoxy-5-ethoxyphenyl; or trisubstitutedphenyl, such as trialkoxy (e.g., 3,4,5-trimethoxy or ethoxyphenyl),3,5-dimethoxy-4-chloro-phenyl, etc.), amino, —SO₂N H₂,—SO₂NH(aliphatic), —SO₂N(aliphatic)₂, —O-aliphatic-COOH, and—O-aliphatic-NH₂ (which may contain one or two N-aliphatic or N-acylsubstituents).

[0111] A “halo” substituent according to the present invention may be afluoro, chloro, bromo or iodo substituent. Fluoro is often the preferredhalogen.

[0112] Compounds of formula II, exclusive of any compounds depicted inTable III, are of special interest and constitute an important class ofnovel compounds. Compounds of this class may differ from rapamycin withrespect to one, two, three, four, five, six or seven substituentmoieties. This class includes among others rapalogs with modifications,relative to rapamycin, at C7 and C13; C7 and C14; C7 and a; C7 and C43;C7 and C24; C7 and C28; C7 and C30; C7, C13 and C14; C7, C13 and; C7,C13 and C43; C7, C13 and C24; C7, C13 and C28; C7, C13 and C30; C7, C14and a; C7, C14 and C43; C7, C14 and C24; C7, C14 and C28; C7, C14 andC30; C7, a and C24; C7, a and C28; C7, a and C30; C7, C24 and C30; C7,C24, C30 and a; C7, C24, C30 and C13; C7, C24, C30 and C14; C24, C30 andC13; C24, C30 and a; C24, C30 and C14; and C24, C30, C13 and a exclusiveof any compounds depicted in Table III or otherwise previously reportedpublicly.

[0113] One subset of improved rapalogs of special interest forpracticing the methods of this invention are those compounds of formulaII (or pharmaceutically acceptable derivatives thereof) in which R^(C7a)is a moiety other than OMe. This subset (“C7 rapalogs”) includescompounds in which one of R^(C7a) and R^(C7b) is H and the other isselected from substituted or unsubstituted alkenyl, aryl, heteroaryl or-Z-aliphatic, Z-aryl, -Z-heteroaryl, or Z-acyl, where Z and Z′ areindependently O, S or NH and acyl comprises —CHO, —(C═O)-aliphatic,—(C═O)-aryl, —(C═O)-heteroaryl, —(C═O)-Z′-aliphatic, —(C═O)-Z′-aryl,—(C═O)-Z′-heteroaryl. In certain embodiments of this subset, R^(C7a) andR^(C7b) are independently selected from the following groups: H; asubstituted or unsubstituted two to eight carbon straightchain, branchedor cyclic alkenyl, alkoxyl or alkylmercapto; and a substituted orunsubstituted aryl, heteroaryl, aryloxy or heteroaryloxy, arylmercaptoor heteroarylmercapto. Compounds of this subset include among othersthose in which R^(C7a) is H; (together with R^(C7b)) ═O; alkoxy;alkylmercapto; amino (1°, 2° or 3°); amido; carbamate; aryl orsubstituted aryl; phenyl or substituted phenyl; substituted orunsubstituted heteroaryl such as substituted or unsubstitutedthiophenyl, furyl, indolyl, etc.; or benzyloxy or substituted benzyloxy.Other illustrative C7 rapalogs and types of C7 rapalogs which may beused in practicing the methods of this invention include those in whichone of R^(C7a) and R^(C7b) is H and the other is selected from —OEt,—O-propyl, —O-butyl, —OCH₂CH₂—OH, —O-benzyl, —O-substituted benzyl(including e.g., 3-nitro-, 4-chloro-, 3-iodo-4-diazo-, 3,4-dimethoxy-,and 2-methoxy-), —S-Me, —S-phenyl, —O(CO)Me, -allyl, —CH₂C(Me)═CH₂,—OCH₂—CCH, —OCH₂—CC-Me, —OCH₂—CC-Et, —OCH₂—CC—CH₂OH, or-2,4-dimethoxyphenyl, 2,4,6-trimethoxyphenyl, furanyl, thiophen-yl,methylthiophen-yl, pyrolyl and indolyl. In the foregoing types ofrapalogs, the hydroxy substituent at C43 may be present in eitherstereochemical orientation or may be modified as described elsewhereherein. C7 rapalogs may further vary from rapamycin at one, two, three,four, five or more other positions as well. C7 rapalogs other than thosedepicted in Table III are novel and are encompassed by this invention ascompositions of matter per se.

[0114] Another subset of improved rapalogs of special interest in thepractice of the various methods of the invention are C30,C24 rapalogs offormula II, i.e., rapalogs of formula II in which R^(C30) and R^(C24)are both other than (═O). Of special interest are those C30,C24 rapalogsin which R^(C7a) is a moiety other than OMe. In certain embodiments ofthis subset, R^(C7a) and R^(C7b) are independently selected from —H,—OR¹, —SR¹, —OC(O)R¹ or —OC(O)NHR¹, —NHR¹, —NHC(O)R¹, —NH—SO₂—R¹ and—R², where R²=substituted aryl or allyl or alkylaryl (e.g. benzyl orsubstituted benzyl), so long as one of R^(C7a) and R^(C7b) is H. Incertain embodiments of this subset, R^(C30) and R^(C24) are both —OH,e.g. in the “S” configuration. In other embodiments R^(C30) and R^(C24)are independently selected from OR³. This subset includes among othersall rapalogs in which R^(C30) and R^(C24) are OH and one of R^(C7a) andR^(C7b) comprises any of the replacement substituents at that positionspecified for formula II, including any of the C7 substituentsidentified in compounds of Tables II or III. This subset includes amongothers rapalogs which differ from rapamycin with respect to the moietya. For instance, this subset includes compounds of the formula:

[0115] where at least one of R^(C7a) and R^(C7b) is other than —OMe.Alternative substituents for R^(C7a) and/or R^(C7b) are as disclosedelsewhere herein. Of special interest are compounds in which one ofR^(C7a) and R^(C7b) is cyclic aliphatic, aryl, heterocyclic orheteroaryl, which may be optionally substituted. Other compounds withinthis subset include those in which one, two, three, four or five of thehydroxyl groups is epimerized, fluorinated, alkylated, acylated orotherwise modified via other ester, carbamate, carbonate or ureaformation. An illustrative compound for example is the compound offormula III in which the hydroxyl group at C43 is epimerized and thehydroxyl groups at C28 and C30 are alkylated, acylated or linked viacarbonate formation. Another subset of improved rapalogs of specialinterest are those compounds of formula II in which one or both ofR^(C13) and R^(C28) is F. In various embodiments of this subset, one,two, three, four or five other substituents in formula II differ fromthe substituents found in rapamycin. For instance, this subset includesC13 fluororapalogs, C28 fluororapalogs and C13, C28-difluororapalogs ofthe following structures, where R^(C7a) and R^(C7b) are as previouslydefined:

[0116] The 13-fluoro rapalogs, including in particular 13-fluororapamycin and analogs and derivatives thereof containing varioussubstituents which do not abolish immunosuppressive activity inrapamycin itself, are of interest as immunosuppressants.

[0117] An interesting intersection of some of the foregoing subsets ofcompounds is the set of improved rapalogs comprising compounds offormula II, or pharmaceutically acceptable derivatives thereof, in whichR^(C24) and R^(C30) are both other than (═O) and one or both of R^(C13)and R^(C28) is F. This set includes, inter alia, 24,30-tetrahydro-13-Frapalogs, 24,30-tetrahydro-28-F rapalogs and 24,30-tetrahydro-13,28-diFrapalogs, as well as C7 variants of any of the foregoing, in whichR^(C7a) is other than OMe. A portion of that set is illustrated by thefollowing structure, where R^(C7a) and R^(C7b) are as previouslydefined:

[0118] These compounds may be further derivatized, e.g., bymodifications at one or both of R^(C14) and R^(C43) relative to the C14and C43 substituents in rapamycin itself.

[0119] Another subset of improved rapalogs of special interest are thosecompounds of formula II in which R^(C14) is other than 0, OH or H, e.g.,compounds wherein R^(C14) is —OR⁶, —NR⁶, —NC(O)R⁶, —OC(O)R⁶ or—OC(O)NR⁶, with or without one or more other modifications relative torapamycin.

[0120] Another subset of improved rapalogs of interest are thosecompounds of formula II in which R^(C13) is other than an alkoxyl groupcomprising a C1-C4 alkyl moiety, with or without one or more othermodifications at other positions relative to rapamycin. For example,this subset includes rapalogs which differ in structure from rapamycinby virtue of possessing (a) in place of OH at C13, a replacementsubstituent R^(C13) which is other than C1-C4 alkoxy, and (b) in placeof MeO at C7, replacement substituents R^(C7a) and R^(C7b) as definedabove.

[0121] Another subset of improved rapalogs of interest are thosecompounds of formula II in which R^(C24) is other than ═O, again, withor without one or more other modifications at other positions relativeto rapamycin.

[0122] Another subset of improved rapalogs which is of special interestin practicing the methods of this invention include those compounds offormula II which share the stereoisomerism of rapamycin and in whichR^(C7a) is —OMe wherein R^(C30) is not ═O, R^(C24) is not ═O, R^(C13) isnot —OH, R^(C14) is not ═O and/or R³ and/or R⁴ are not H.

[0123] Other improved rapalogs of interest include compounds of formulaII in which R^(C14) is OH.

[0124] Furthermore, this invention encompasses improved rapalogs inwhich one or more of the carbon-carbon double bonds at the 1, 2, 3, 4 or5, 6 positions in rapamycin are saturated, alone or in combination witha modification elsewhere in the molecule, e.g. at one or more of C7,C13, C43, C24 C28 and/or C30. It should also be appreciated that theC3,C4 double bond may be epoxidized; that the C6 methyl group may bereplaced with —CH₂OH or —CH₂OMe; that the C43 hydroxy may be convertedto F, Cl or H or other substituent; and that the C42 methoxy moiety maybe demethylated, in any of the compounds disclosed herein, using methodsknown in the art. Likewise, moiety “a” may be replaced with any of thefollowing

[0125] Synthetic Guidance

[0126] The production of rapamycin by fermentation and by totalsynthesis is known. The production of a number of rapalogs asfermentation products is also known. These include among others rapalogsbearing alternative moieties to the characteristic cyclohexyl ring orpipecolate ring of rapamycin, as well as C7-desmethyl-rapamycin,C29-desmethyl-rapamycin and C29-desmethoxyrapamycin.

[0127] Methods and materials for effecting various chemicaltransformations of rapamycin and structurally related macrolides areknown in the art, as are methods for obtaining rapamycin and variousrapalogs by fermentation. Many such chemical transformations ofrapamycin and various rapalogs are disclosed in the patent documentsidentified in Table I, above, which serve to illustrate the level ofskill and knowledge in the art of chemical synthesis and productrecovery, purification and formulation which may be applied inpracticing the subject invention. The following representativetransformations and/or references which can be employed to produce thedesired rapalogs are illustrative: ring position modified literaturereference C7 Luengo, et al. JOC 59, 6512 (1995); Chem & Biol 2(7),471-481 (1995) C-13 C13—>F: protect C28 and C43, rxn at 0° C-14Schubert, et al. Angew Chem Int Ed Engl 23, 167 (1984). C-20 Nelson, USPatent 5,387,680 C-24 US Patent 5,373,014; 5,378,836 Lane, et al.Synthesis 1975, p136. C-30 Luengo et al. Tet. Lett. 35, 6469 (1994)various Or et al, U.S. Pat. Nos. 5,527,907 and 5,583,139 positionsLuengo, WO 94/02136; Cottens et al, WO 95/16691

[0128] Approaches to the synthesis of the various fluoro and difluororapalogs are presented schematically below:

[0129] An approach to the synthesis of various 24,30-tetrahydro rapalogsis illustrated below:

[0130] By way of further example, starting with 13-fluoro rapamycininstead of rapamycin yields the corresponding 13-fluoro-24,30-tetrahydroC7 rapalog.

[0131] One approach for the synthesis of other C13 derivatives isillustrated below:

[0132] Additionally, it is contemplated that rapalogs for use in thisinvention as well as intermediates for the production of such rapalogsmay be prepared by directed biosynthesis, e.g. as described by Katz etal, WO 93/13663 and by Cane et al, WO 9702358.

[0133] Novel rapalogs of this invention may be prepared by one ofordinary skill in this art relying upon methods and materials known inthe art as guided by the disclosure presented herein. For instance,methods and materials may be adapted from known methods set forth orreferenced in the documents cited above, the full contents of which areincorporated herein by reference. Additional guidance and examples areprovided herein by way of illustration and further guidance to thepractitioner. It should be understood that the chemist of ordinary skillin this art would be readily able to make modifications to theforegoing, e.g. to add appropriate protecting groups to sensitivemoieties during synthesis, followed by removal of the protecting groupswhen no longer needed or desired, and would be readily capable ofdetermining other synthetic approaches.

[0134] FKBP Domains and Fusion Proteins

[0135] The FKBP fusion protein comprises at least one FKBP domaincontaining all or part of the peptide sequence of an FKBP domain and atleast one heterologous action domain. This chimeric protein must becapable of binding to an improved rapalog of this invention, preferablywith a Kd value below about 100 nM, more preferably below about 10 nMand even more preferably below about 1 nM, as measured by direct bindingmeasurement (e.g. fluorescence quenching), competition bindingmeasurement (e.g. versus FK506), inhibition of FKBP enzyme activity(rotamase), or other assay methodology. Typically the chimeric proteinwill contain one or more protein domains comprising peptide sequenceselected from that of a naturally occurring FKBP protein such as humanFKBP12, e.g. as described in International Patent ApplicationPCT/US94/01617. That peptide sequence may be modified to adjust thebinding specificity, usually with replacement, insertion or deletion of10 or fewer, preferably 5 or fewer, amino acid residues. Suchmodifications are elected in certain embodiments to yield one or both ofthe following binding profiles: (a) binding of an improved rapalog tothe modified FKBP domain, or chimera containing it, preferably at leastone, and more preferably at least two, and even more preferably three orfour or more, orders of magnitude better (by any measure) than to FKBP12or the FKBP endogenous to the host cells to be engineered; and (b)binding of the FKBP:rapalog complex to the FRB fusion protein,preferably at least one, and more preferably at least two, and even morepreferably at least three, orders of magnitude better (by any measure)than to the FRAP or other FRB-containing protein endogenous to the hostcell to be engineered.

[0136] The FKBP chimera also contains at least one heterologous actiondomain, i.e., a protein domain containing non-FKBP peptide sequence. Theaction domain may be a DNA-binding domain, transcription activationdomain, cellular localization domain, intracellular signal transductiondomain, etc., e.g. as described elsewhere herein or in PCT/US94/01617 orthe other cited references. Generally speaking, the action domain iscapable of directing the chimeric protein to a selected cellularlocation or of initiating a biological effect upon association oraggregation with another action domain, for instance, uponmultimerization of proteins containing the same or different actiondomains.

[0137] A recombinant nucleic acid encoding such a fusion protein will becapable of selectively hybridizing to a DNA encoding the parent FKBPprotein, e.g. human FKBP12, or would be capable of such hybridizationbut for the degeneracy of the genetic code. Since these chimericproteins contain an action domain derived from another protein, e.g.Gal4, VP16, FAS, CD3 zeta chain, etc., the recombinant DNA encoding thechimeric protein will also be capable of selectively hybridizing to aDNA encoding that other protein, or would be capable of suchhybridization but for the degeneracy of the genetic code.

[0138] FKBP fusion proteins of this invention, as well as FRB fusionproteins discussed in further detail below, may contain one or morecopies of one or more different ligand binding domains and one or morecopies of one or more action domains. The ligand binding domain(s)(i.e., FKBP and FRB domains) may be N-terminal, C-terminal, orinterspersed with respect to the action domain(s). Embodiments involvingmultiple copies of a ligand binding domain usually have 2, 3 or 4 suchcopies. For example, an FKBP fusion protein may contain 2, 3 or 4 FKBPdomains. The various domains of the FKBP fusion proteins (and of the FRBfusion proteins discussed below) are optionally separated by linkingpeptide regions which may be derived from one of the adjacent domains ormay be heterologous.

[0139] Illustrative examples of FKBP fusion proteins useful in thepractice of this invention include the FKBP fusion proteins disclosed inPCT/US94/01617 (Stanford & Harvard), PCT/US94/08008 (Stanford &Harvard), Spencer et al (supra), PCT/US95/10591 (ARIAD), PCT/US95/06722(Mitotix, Inc.) and other references cited herein; the FKBP fusionproteins disclosed in the examples which follow; variants of any of theforegoing FKBP fusion proteins which contain up to 10 (preferably 1-5)amino acid insertions, deletions or substitutions in one or more of theFKBP domains and which are still capable of binding to rapamycin or to arapalog; variants of any of the foregoing FKBP fusion proteins whichcontain one or more copies of an FKBP domain which is encoded by a DNAsequence capable of selectively hybridizing to a DNA sequence encoding anaturally occurring FKBP domain and which are still capable of bindingto rapamycin or to a rapalog; variants of any of the foregoing in whichone or more heterologous action domains are deleted, replaced orsupplemented with a different heterologous action domain; variants ofany of the foregoing FKBP fusion proteins which are capable of bindingto rapamycin or a rapalog and which contain an FKBP domain derived froma non-human source; and variants of any of the foregoing FKBP fusionproteins which contain one or more amino acid residues corresponding toTyr26, Phe36, Asp37, Arg42, Phe46, Phe48, Glu54, Val55, or Phe99 ofhuman FKBP12 in which one or more of those amino acid residues isreplaced by a different amino acid, the variant being capable of bindingto rapamycin or a rapalog.

[0140] For instance, in a number of cases the FKBP fusion proteinscomprise multiple copies of an FKBP domain containing amino acids 1-107of human FKBP12, separated by the 2-amino acid linker Thr-Arg encoded byACTAGA, the ligation product of DNAs digested respectively with therestriction endonucleases SpeI and XbaI. The following table providesillustrative subsets of mutant FKBP domains based on the foregoingFKBP12 sequence:

Illustrative Mutant FKBPs

[0141] F36A Y26V F46A W59A F36V Y26S F48H H87W F36M D37A F48L H87R F36SI90A F48A F36V/F99A F99A I91A E54A F36V/F99G F99G F46H E54K F36M/F99AY26A F46L V55A F36M/F99G

[0142] note: Entries identify the native amino acid by single lettercode and sequence position, followed by the replacement amino acid inthe mutant. Thus, F36V designates a human FKBP12 sequence in whichphenylalanine at position 36 is replaced by valine. F36V/F99A indicatesa double mutation in which phenylalanine at positions 36 and 99 arereplaced by valine and alanine, respectively.

[0143] FRB Domains and Fusion Proteins

[0144] The FRB fusion protein comprises at least one FRB domain (whichmay comprise all or part of the peptide sequence of a FRAP protein or avariant thereof, as described elsewhere) and at least one heterologouseffector domain.

[0145] Generally speaking, the FRB domain, or a chimeric proteinencompassing it, is encoded by a DNA molecule capable of hybridizingselectively to a DNA molecule encoding a protein comprising a naturallyoccurring FRB domain, e.g. a DNA molecule encoding a human or othermammalian FRAP protein or one of yeast proteins, Tor-1 or Tor-2 or thepreviously mentioned Candida FRB-containing protein. FRB domains of thisinvention include those which are capable of binding to a complex of anFKBP protein and an improved rapalog of this invention.

[0146] The FRB fusion protein must be capable of binding to the complexformed by the FKBP fusion protein with an improved rapalog of thisinvention. Preferably, the FRB fusion protein binds to that complex witha Kd value below 200 μM, more preferably below 10 μM, as measured byconventional methods. The FRB domain will be of sufficient length andcomposition to maintain high affinity for a complex of the rapalog withthe FKBP fusion protein. In some embodiments the FRB domain spans fewerthan about 150 amino acids in length, and in some cases fewer than about100 amino acids. One such region comprises a 133 amino acid region ofhuman FRAP extending from Val2012 through Tyr2144. See Chiu et al, 1994,Proc. Natl. Acad. Sci. USA 91:12574-12578. An FRB region of particularinterest spans Glu2025 through Gln2114 of human FRAP and retainsaffinity for a FKBP12-rapamycin complex or for FKBP-rapalog complex. Insome embodiments Q2214 is removed from the 90-amino acid sequencerendering this an 89-amino acid FRB domain. The FRB peptide sequence maybe modified to adjust the binding specificity, usually with replacement,insertion or deletion, of 10 or fewer, preferably 5 or fewer, aminoacids. Such modifications are elected in certain embodiments to achievea preference towards formation of the complex comprising one or moremolecules of the FKBP fusion protein, FRB fusion protein and an improvedrapalog over formation of complexes of endogenous FKBP and FRAP proteinswith the rapalog. Preferably that preference is at least one, and morepreferably at least two, and even more preferably three, orders ofmagnitude (by any measure).

[0147] A recombinant DNA encoding such a protein will be capable ofselectively hybridizing to a DNA encoding a FRAP species, or would becapable of such hybridization but for the degeneracy of the geneticcode. Again, since these chimeric proteins contain an effector domainderived from another protein, e.g. Gal4, VP16, Fas, CD3 zeta chain,etc., the recombinant DNA encoding the chimeric protein will be capableof selectively hybridizing to a DNA encoding that other protein, orwould be capable of such hybridization but for the degeneracy of thegenetic code.

[0148] Illustrative examples of FRB chimeras useful in the practice ofthis invention include those disclosed in the examples which follow,variants thereof in which one or more of the heterologous domains arereplaced with alternative heterologous domains or supplemented with oneor more additional heterologous domains, variants in which one or moreof the FRB domains is a domain of non-human peptide sequence origin(such as Tor 2 or Candida for example), and variants in which the FRBdomain is modified by amino acid substitution, replacement or insertionas described herein, so long as the chimera is capable of binding to acomplex formed by an FKBP protein and an improved rapalog of thisinvention. An illustrative FRB fusion protein contains one or more FRBsof at least 89-amino acids, containing a sequence spanning at leastresidues 2025-2113 of human FRAP, separated by the linker Thr-Arg formedby ligation of SpeI-XbaI sites as mentioned previously. It should beappreciated that such restriction sites or linkers in any of the fusionproteins of this invention may be deleted, replaced or extended usingconventional techniques such as site-directed mutagenesis.

[0149] Mixed Chimeric Proteins

[0150] A third type of chimeric protein comprises one or more FKBPdomains, one or more heterologous effector domains, and one or more FRBdomains as described for the FRB fusion proteins.

[0151] Mixed chimeric protein molecules are capable of forminghomodimeric or homomultimeric protein complexes in the presence of animproved rapalog to which they bind. Embodiments involving mixedchimeras have the advantage of requiring the introduction into cells ofa single recombinant nucleic acid construct in place of two recombinantnucleic acid constructs otherwise required to direct the expression ofboth an FKBP fusion protein and a FRB fusion protein.

[0152] A recombinant DNA encoding a mixed chimeric protein will becapable of selectively hybridizing to a DNA encoding an FKBP protein, aDNA encoding FRAP, and a heterologous DNA sequence encoding the proteinfrom which one or more effector domains is derived (e.g. Gal4, VP16,Fas, CD3 zeta chain, etc.), or would be capable of such hybridizationbut for the degeneracy of the genetic code.

[0153] Heterologous Domains

[0154] As mentioned above, the heterologous effector domains of the FKBPand FRB fusion proteins are protein domains which, upon mutualassociation of the chimeric proteins bearing them, are capable oftriggering (or inhibiting) DNA-binding and/or transcription of a targetgene; actuating cell growth, differentiation, proliferation orapoptosis; directing proteins to a particular celllular location; oractuating other biological events.

[0155] Embodiments involving regulatable gene transcription involve theuse of target gene constructs which comprise a target gene (whichencodes a polypeptide, antisense RNA, ribozyme, etc. of interest) underthe transcriptional control of a DNA element responsive to theassociation or multimerization of the heterologous domains of the 1stand 2d chimeric proteins.

[0156] In embodiments of the invention involving direct activation oftranscription, the heterologous domains of the 1st and 2d chimericproteins comprise a DNA binding domain such as Gal4 or a chimeric DNAbinding domain such as ZFHD1, discussed below, and a transcriptionalactivating domain such as those derived from VP16 or p65, respectively.The multimerization of a chimeric protein containing such atranscriptional activating domain to a chimeric protein containing a DNAbinding domain targets the transcriptional activator to the promoterelement to which the DNA binding domain binds, and thus activates thetranscription of a target gene linked to that promoter element.Foregoing the transcription activation domain or substituting arepressor domain (see PCT/US94/01617) in place of a transcriptionactivation domain provides an analogous chimera useful for inhibitingtranscription of a target gene. Composite DNA binding domains and DNAsequences to which they bind are disclosed in Pomerantz et al, 1995,supra, the contents of which are incorporated herein by reference. Suchcomposite DNA binding domains may be used as DNA binding domains in thepractice of this invention, together with a target gene constructcontaining the cognate DNA sequences to which the composite DBD binds.

[0157] In embodiments involving indirect activation of transcription,the heterologous domains of the chimeras are effector domains ofsignaling proteins which upon aggregation or multimerization trigger theactivation of transcription under the control of a responsive promoter.For example, the signaling domain may be the intracellular domain of thezeta subunit of the T cell receptor, which upon aggregation, triggerstranscription of a gene linked to the IL-2 promoter or a derivativethereof (e.g. iterated NF-AT binding sites).

[0158] In another aspect of the invention, the heterologous domains areprotein domains which upon mutual association are capable of triggeringcell death. Examples of such domains are the intracellular domains ofthe Fas antigen or of the TNF R1. Chimeric proteins containing a Fasdomain can be designed and prepared by analogy to the disclosure ofPCT/US94/01617.

[0159] Engineered Receptor Domains

[0160] As noted previously, the FKBP and FRB domains may contain peptidesequence selected from the peptide sequences of naturally occurring FKBPand FRB domains. Naturally occurring sequences include those of humanFKBP12 and the FRB domain of human FRAP. Alternatively, the peptidesequences may be derived from such naturally occurring peptide sequencesbut contain generally up to 10, and preferably 1-5, mutations in one orboth such peptide sequences. As disclosed in greater detail elswhereherein, such mutations can confer a number of important features. Forinstance, an FKBP domain may be modified such that it is capable ofbinding an improved rapalog preferentially, i.e. at least one,preferably two, and even more preferably three or four or more orders ofmagnitude more effectively, with respect to rapalog binding by theunmodified FKBP domain. An FRB domain may be modified such that it iscapable of binding a (modified or unmodified) FKBP:rapalog complexpreferentially, i.e. at least one, preferably two, and even morepreferably three orders of magnitude more effectively, with respect tothe unmodified FRB domain. FKBP and FRB domains may be modified suchthat they are capable of forming a tripartite complex with an improvedrapalog, preferentially, i.e. at least one, preferably two, and evenmore preferably three orders of magnitude more effectively, with respectto unmodified FKBP and FRB domains.

[0161] (a) FKBP

[0162] Methods for identifying FKBP mutations that confer enhancedability to bind derivatives of FK506 containing various substituents(“bumps”) were disclosed in PCT/US94/01617. Similar strategies can beused to obtain modified FKBPs that preferentially bind bumped rapamycinderivatives, i.e., rapalogs. The structure of the complex betweenrapamycin and FKBP12 is known (see for example Van Duyne et al., J. Am.Chem. Soc. (1991) 113, 7433-7434). Such data can be used to reveal aminoacid residues that would clash with various rapalog substituents. Inthis approach, molecular modelling is used to identify candidate aminoacid substitutions in the FKBP domain that would accommodate the rapalogsubstituent(s), and site-directed mutagenesis may then be used toengineer the protein mutations so identified. The mutants are expressedby standard methods and their binding affinity for the rapalogsmeasured, for example by inhibition of rotamase activity, or bycompetition for binding with a molecule such as FK506, if the mutantretains appropriate activity/affinity.

[0163] More particularly, we contemplate that certain improved rapalogsof this invention, e.g. rapalogs with modifications relative torapamycin at C-13 or C-14 bind preferentially to FKBPs in which one ormore of the residues, Tyr26, Phe36, Asp37, Tyr82 and Phe99, aresubstituted with amino acids that have smaller side chains (such as Gly,Ala, Val, Met and Ser). Examples of mutant FKBPs with modifications atpositions 26 or 36 are noted in the “Illustrative Mutant FKBPs” tableabove. Similarly, we contemplate that rapalogs with modifications at C20(i.e., rapalogs in which R4 is other than —H) bind preferentially toFKBPs in which Tyr82 and/or Ile56 are replaced by other amino acids,especially those with smaller side chains. In a further example, wecontemplate that rapalogs bearing modifications at C24 (i.e., in which Wis other than ═O) bind preferentially to FKBPs in which one or more ofPhe46, Phe48 and Val55 are replaced by other amino acids, againespecially those with smaller side chains. Moreover, we envisage thatrapalogs with modifications at C28 and/or C30 (i.e., in which R3 isother than H and/or V is other than ═O) bind preferentially to FKBPs inwhich Glu54 is replaced by another amino acid, especially one with asmaller side chain. In all of the above examples, single or multipleamino acid substitutions may be made. Again, specific examples are notedin the previous table.

[0164] An alternative to iterative engineering and testing of single ormultiple mutants is to co-randomize structurally-identified residuesthat are or would be in contact with or near one or more rapalog orrapamycin substituents. A collection of polypeptides containing FKBPdomains randomized at the identified positions (such as are noted in theforegoing paragraph) is prepared e.g. using conventional synthetic orgenetic methods. Such a collection represents a set of FKBP domainscontaining replacement amino acids at one or more of such positions. Thecollection is screened and FKBP variants are selected which possess thedesired rapalog binding properties. In general, randomizing severalresidues simultaneously is expected to yield compensating mutants ofhigher affinity and specificity for a given bumped rapalog as itmaximizes the likelihood of beneficial cooperative interactions betweensidechains. Techniques for preparing libraries randomized at discretepositions are known and include primer-directed mutagenesis usingdegenerate oligonucleotides, PCR with degenerate oligonucleotides, andcassette mutagenesis with degenerate oligonucleotides (see for exampleLowman, H. B, and Wells, J. A. Methods: Comp. Methods Enzymol. 1991. 3,205-216; Dennis, M. S. and Lazarus, R. A. 1994. J. Biol. Chem. 269,22129-22136; and references therein).

[0165] We further contemplate that in many cases, randomization of onlythe few residues in or near direct contact with a given position inrapamycin may not completely explore all the possible variations in FKBPconformation that could optimally accommodate a rapalog substituent(bump). Thus the construction is also envisaged of unbiased librariescontaining random substitutions that are not based on structuralconsiderations, to identify subtle mutations or combinations thereofthat confer preferential binding to bumped rapalogs. Several suitablemutagenesis schemes have been described, including alanine-scanningmutagenesis (Cunningham and Wells (1989) Science 244, 1081-1085), PCRmisincorporation mutagenesis (see eg. Cadwell and Joyce, 1992, PCR Meth.Applic. 2, 28-33), and ‘DNA shuffling’ (Stemmer, 1994, Nature 370,389-391 and Crameri et al, 1996, Nature Medicine 2, 100-103). Thesetechniques produce libraries of random mutants, or sets of singlemutants, that are then searched by screening or selection approaches.

[0166] In many cases, an effective strategy to identify the best mutantsfor preferential binding of a given bump is a combination ofstructure-based and unbiased approaches. See Clackson and Wells, 1994,Trends Biotechnology 12, 173-184 (review). For example we contemplatethe construction of libraries in which key contact residues arerandomized by PCR with degenerate oligonucleotides, but withamplification performed using error-promoting conditions to introducefurther mutations at random sites. A further example is the combinationof component DNA fragments from structure-based and unbiased randomlibraries using DNA shuffling. Screening of libraries for desirablemutations may be performed by use of a yeast 2-hybrid system (Fields andSong (1989) Nature 340, 245-246). For example, an FRB-VP16 fusion may beintroduced into one vector, and a library of randomized FKBP sequencescloned into a separate GAL4 fusion vector. Yeast co-transformants aretreated with rapalog, and those harboring complementary FKBP mutants areidentified by for example beta-galactosidase or luciferase production (ascreen), or survival on plates lacking an essential nutrient (aselection), as appropriate for the vectors used. The requirement forbumped rapamycin to bridge the FKBP-FRAP interaction is a useful screento eliminate false positives.

[0167] A further strategy for isolating modified ligand-binding domainsfrom libraries of FKBP (or FRB) mutants utilizes a genetic selection forfunctional dimer formation described by Hu et. al. (Hu, J. C., et al.1990. Science. 250:1400-1403; for review see Hu, J. C. 1995. Structure.3:431-433). This strategy utilizes the fact that the bacteriophagelambda repressor cI binds to DNA as a homodimer and that binding of suchhomodimers to operator DNA prevents transcription of phage genesinvolved in the lytic pathway of the phage life cycle. Thus, bacterialcells expressing functional lambda repressor are immune to lysis bysuperinfecting phage lambda. Repressor protein comprises an aminoterminal DNA binding domain (amino acids 1-92), joined by a 40 aminoacid flexible linker to a carboxy terminal dimerization domain. Theisolated N-terminal domain binds to DNA with low affinity due toinefficient dimer formation. High affinity DNA binding can be restoredwith heterologous dimerization domains such as the GCN4 “leucinezipper”. Hu et al have described a system in which phage immunity isused as a genetic selection to isolate GCN4 leucine zipper mutantscapable of mediating lambda repressor dimer formation from a largepopulation of sequences (Hu et. al., 1990).

[0168] For example, to use the lambda repressor system to identify FRAPmutants complementary to bumped rapalogs, lambda repressor-FRAPlibraries bearing mutant FRAP sequences are transformed into E. colicells expressing wildtype lambda repressor-FKBP protein. Plasmidsexpressing FRAP mutants are isolated from those colonies that survivelysis on bacterial plates containing high titres of lambda phage and“bumped” rapamycin compounds. Alternatively, to isolate FKBP mutants,the above strategy is repeated with lambda repressor-FKBP librariesbearing mutant FKBP sequences transformed into E. coli cells expressingwildtype lambda repressor-FRAP protein.

[0169] A further alternative is to clone the randomized FKBP sequencesinto a vector for phage display, allowing in vitro selection of thevariants that bind best to the rapalog. Affinity selection in vitro maybe performed in a number of ways. For example, rapalog is mixed with thelibrary phage pool in solution in the presence of recombinant FRAPtagged with an affinity handle (for example a hexa-histidine tag, orGST), and the resultant complexes are captured on the appropriateaffinity matrix to enrich for phage displaying FKBP harboringcomplementary mutations. Techniques for phage display have beendescribed, and other in vitro selection selection systems can also becontemplated (for example display on lambda phage, display on plasmids,display on baculovirus). Furthermore, selection and screening strategiescan also be used to improve other properties of benefit in theapplication of this invention, such as enhanced stability in vivo. For areview see Clackson, T. & Wells, J. A. 1994. Trends Biotechnol. 12,173-184.

[0170] (b) FRAP

[0171] Similar considerations apply to the generation of mutant FRBdomains which bind preferentially to improved rapalogs containingmodifications (i.e., are ‘bumped’) relative to rapamycin in theFRAP-binding portion of the macrocycle. For example, one may obtainpreferential binding using rapalogs bearing substituents other than —OMeat the C7 position with FRBs based on the human FRAP FRB peptidesequence but bearing amino acid substitutions for one of more of theresidues Tyr2038, Phe2039, Thr2098, Gln2099, Trp2101 and Asp2102.Exemplary mutations include Y2038H, Y2038L, Y2038V, Y2038A, F2039H,F2039L, F2039A, F2039V, D2102A, T2098A, T2098N, and T2098S. Rapalogsbearing substituents other than —OH at C28 and/or substituents otherthan ═O at C30 may be used to obtain preferential binding to FRAPproteins bearing an amino acid substitution for Glu2032. Examplarymutations include E2032A and E2032S. Proteins comprising an FRBcontaining one or more amino acid replacements at the foregoingpositions, libraries of proteins or peptides randomized at thosepositions (i.e., containing various substituted amino acids at thoseresidues), libraries randomizing the entire protein domain, orcombinations of these sets of mutants are made using the proceduresdescribed above to identify mutant FRAPs that bind preferentially tobumped rapalogs.

[0172] The affinity of candidate mutant FRBs for the complex of an FKBPprotein complexed with a rapalog may be assayed by a number oftechniques; for example binding of in vitro translated FRB mutants toGST-FKBP in the presence of drug (Chen et al. 1995. Proc. Natl. Acad.Sci. USA 92, 4947-4951); or ability to participate in arapalog-dependent transcriptionally active complex with an appropriateFKBP fusion protein in a yeast two-hybrid assay.

[0173] FRB mutants with desired binding properties may be isolated fromlibraries displayed on phage using a variety of sorting strategies. Forexample, a rapalog is mixed with the library phage pool in solution inthe presence of recombinant FKBP tagged with an affinity handle (forexample a hexa-histidine tag, or GST), and the resultant complexes arecaptured on the appropriate affinity matrix to enrich for phagedisplaying FRAP harboring complementary mutations.

[0174] An additional feature of the FRB fusion protein that may vary inthe various embodiments of this invention is the exact sequence of theFRB domain used. In some applications it may be preferred to useportions of an FRB which are larger than the minimal (89 amino acid) FRBdomain. These include extensions N-terminal to residue Glu2025(preferably extending to at least Arg2018 or Ile2021), as well asC-terminal extensions beyond position 2113, e.g. to position 2113, 2141or 2174 or beyond), which may in some cases improve the stability of thefolded FRB domain and/or the efficiency of expression. Otherapplications in which different FRB sequence termini may be used includethose in which a long linker is desired for steric reasons on one orboth sides of the FRB domain, for example to accommodate the distortionsof the polypeptide chain required for FRB-mediated protein-proteinassociation at the cell membrane or on DNA. Conversely, in otherapplications short linkers on one or both sides of the FRB domain may bepreferred or required to present the heterologous effector domain(s)appropriately for biological function. In human gene therapyapplications the use of naturally occurring human FRAP sequence for suchlinkers will generally be preferred to the introduction of heterologoussequences, or reduce the risk of provoking an immune response in thehost organism.

[0175] Some rapalogs, especially rapalogs with modifications orsubstituents (relative to rapamycin) at positions believed to lie nearthe boundary between the FKBP binding domain and the FRAP bindingdomain, such as those on C28, C30, C7 and C24, possess reduced ability,relative to rapamycin, to form complexes with both mammalian FKBP andFRB domains, in particular, with those domains containing naturallyoccurring human peptide sequence. That reduced ability may be manifestedas a reduced binding affinity as determined by any of the direct orindirect assay means mentioned herein or as reduced immunosuppressiveactivity as determined in an appropriate assay such as a T cellproliferation assay. In such cases, iterative procedures may be used toidentify pairs of mutant FKBPs and mutant FRBs that are capable ofcomplexing with the rapalog more effectively than the correspondingdomains containing naturally occurring human peptide sequence. Forexample, one may first identify a complementary modified FKBP domaincapable of binding to the rapalog, as discussed previously, and thenusing this mutant FKBP domain as an affinity matrix in complex with therapalog, one may select a complementary modified FRB domain capable ofassociating with that complex. Several cycles of such mutagenesis andscreening may be performed to optimize the protein pair.

[0176] For some embodiments, it will be desirable to use FRB and/or FKBPdomains containing mutations that can affect the protein-proteininteraction. For instance, mutant FKBP domains which when bound to agiven rapalog are capable of complexing with an endogenous FRBmeasurably less effectively than to a mutant FRB are of particularinterest. Also of interest are mutant FRB domains which are capable ofassociating with a complex of a mutant FKBP with a given rapalogmeasurable more effectively than with a complex of an endogenous FKBPwith the rapalog. Similar selection and screening approaches to thosedelineated previously can be used (i) to identify amino acidsubstitutions, deletions or insertions to an FKBP domain whichmeasurably diminish the domain's ability to form the tripartite complexwith a given rapalog and the endogenous FRB; (ii) to identify amino acidsubstitutions, deletions or insertions to an FRB domain which measurablydiminish the domain's ability to form the tripartite complex with agiven rapalog and the endogenous FKBP; and (iii) to select and/orotherwise identify compensating mutation(s) in the partner protein. Asexamples of suitable mutant FKBPs with diminished effectiveness intripartite complex formation, we include mammalian, preferably humanFKBP in which one or both of His87 and Ile90 are replaced with aminoacids such as Arg, Trp, Phe, Tyr or Lys which contain bulky side chaingroups; FRB domains, preferably containing mammalian, and morepreferably of human, peptide sequence may then be mutated as describedabove to generate complementary variants which are capable of forming atripartite complex with the mutant FKBP and a given rapalog.Illustrative FRB mutations which may be useful with H87W or H87RhFKBP12s include human FRBs in which Y2038 is replaced by V, S, A or L;F2039 is replaced by A; and/or R2042 is replaced by L, A or S.Illustrative FRB mutations which may be useful with 190W or 190RhFKBP12s include human FRBs in which K2095 is replaced with L, S, A orT.

[0177] Additionally, in optimizing the receptor domains of thisinvention, it should be appreciated that immunogenicity of a polypeptidesequence is thought to require the binding of peptides by MHC proteinsand the recognition of the presented peptides as foreign by endogenousT-cell receptors. It may be preferable, at least in human gene therapyapplications, to tailor a given foreign peptide sequence, includingjunction peptide sequences, to minimize the probability of its beingimmunologically presented in humans. For example, peptide binding tohuman MHC class I molecules has strict requirements for certain residuesat key ‘anchor’ positions in the bound peptide: eg. HLA-A2 requiresleucine, methionine or isoleucine at position 2 and leucine or valine atthe C-terminus (for review see Stern and Wiley (1994) Structure 2,145-251). Thus in engineering proteins in the practice of thisinvention, this periodicity of these residues is preferably avoided,especially in human gene therapy applications. The foregoing applies toall protein engineering aspects of the invention, including withoutlimitation the engineering of point mutations into receptor domains, andto the choice or design of boundaries between the various proteindomains.

[0178] Other Components, Design Features and Applications

[0179] The chimeric proteins may contain as a heterologous domain acellular localization domain such as a membrane retention domain. Seee.g. PCT/US94/01617, especially pages 26-27. Briefly, a membraneretention domain can be isolated from any convenient membrane-boundprotein, whether endogenous to the host cell or not. The membraneretention domain may be a transmembrane retention domain, i.e., an aminoacid sequence which extends across the membrane as in the case of cellsurface proteins, incluing many receptors. The transmembrane peptidesequence may be extended to span part or all of an extracellular and/orintracellular domain as well. Alternatively, the membrane retentiondomain may be a lipid membrane retention domain such as a myristoylationor palmitoylation site which permits association with the lipids of thecell surface membrane. Lipid membrane retention domains will usually beadded at the 5′ end of the coding sequence for N-terminal binding to themembrane and, proximal to the 3′ end for C-terminal binding. Peptidesequences involving post-translational processing to provide for lipidmembrane binding are described by Carr, et al., PNAS USA (1988) 79,6128; Aitken, et al., FEBS Lett. (1982) 150, 314; Henderson, et al.,PNAS USA (1983) 80, 319; Schulz, et al., Virology (1984), 123, 2131;Dellman, et al., Nature (1985) 314, 374; and reviewed in Ann. Rev. ofBiochem. (1988) 57, 69. An amino acid sequence of interest includes thesequence M-G-S-S-K-S-K-P-K-D-P-S-Q-R. Various DNA sequences can be usedto encode such sequences in the various chimeric proteins of thisinvention. Other localization domains include organelle-targetingdomains and sequences such as -K-D-E-L and -H-D-E-L which targetproteins bearing them to the endoplasmic reticulum, as well as nuclearlocalization sequences which are particularly useful for chimericproteins designed for (direct) transcriptional regulation. Variouscellular localization sequences and signals are well known in the art.

[0180] Further details which may be used in the practice of the subjectinvention relating to the design, assembly and use of constructsencoding chimeric proteins containing various effector domains includingcytoplasmic signal initiation domains such as the CD3 zeta chain,nuclear transcription factor domains including among others VP16 andGAL4, domains capable of triggering apoptosis including the Fascytoplasmic domain and others are disclosed in PCT/US94/01617 andPCT/US95/10591. The latter international application further disclosesadditional features particularly applicable to the creation ofgenetically engineered animals which may be used as disease models inbiopharmaceutical research. Those features include the use of tissuespecific regulatory elements in the constructs for expression of thechimeric proteins and the application of regulated transcription to theexpression of Cre recombinase as the target gene leading to theelimination of a gene of interest flanked by loxP sequences.Alternatively, flp and its cognate recognition sequences may be usedinstead of Cre and lox. Those features may be adapted to the subjectinvention.

[0181] In various cases, especially in embodiments involving wholeanimals containing cells engineered in accordance with this invention,it will often be preferred, and in some cases required, that the variousdomains of the chimeric proteins be derived from proteins of the samespecies as the host cell. Thus, for genetic engineering of human cells,it is often preferred that the heterologous domains (as well as the FKBPand FRB domains) be of human origin, rather than of bacterial, yeast orother non-human source.

[0182] We also note that epitope tags may also be incorporated intochimeric proteins of this invention to permit convenient detection.

[0183] Tissue-Specific or Cell-Type Specific Expression

[0184] It will be preferred in certain embodiments, that the chimericproteins be expressed in a cell-specific or tissue-specific manner. Suchspecificity of expression may be achieved by operably linking one oremore of the DNA sequences encoding the chimeric protein(s) to acell-type specific transcriptional regulatory sequence (e.g.promoter/enhancer). Numerous cell-type specific transcriptionalregulatory sequences are known. Others may be obtained from genes whichare expressed in a cell-specific manner. See e.g. PCT/US95/10591,especially pp. 36-37.

[0185] For example, constructs for expressing the chimeric proteins maycontain regulatory sequences derived from known genes for specificexpression in selected tissues.

[0186] Representative examples are tabulated below: Tissue GeneReference lens g2-crystallin Breitman, M. L., Clapoff, S., Rossant, J.,Tsui, L. C., Golde, L. M., Maxwell, I. H., Bernstin, A. (1987) GeneticAblation: targeted expression of a toxin gene causes microphthalmia intransgenic mice. Science 238: 1563-1565 aA-crystallin Landel, C. P.,Zhao, J., Bok, D., Evans, G. A. (1988) Lens-specific expression of arecombinant ricin induces developmental defects in the eyes oftransgenic mice. Genes Dev. 2: 1168-1178 Kaur, S., key, B., Stock, J.,McNeish, J. D., Akeson, R., Potter, S. S. (1989) Targeted ablation ofalpha-crystallin-synthesizing cells produces lens-deficient eyes intransgenic mice. Development 105: 613-619 pituitary - Growth hormoneBehringer, R. R., Mathews, L. S., Palmiter, R. D., Brinster, R. L.somatrophic (1988) Dwarf mice produced by genetic ablation of growthcells hormone-expressing cells. Genes Dev. 2: 453-461. pancreas Insulin-Ornitz, D. M., Palmiter, R. D., Hammer, R. E., Brinster, R. L.,Elastase - acinar Swift, G. H., MacDonald, R. J. (1985) Specificexpression of an cell specific elastase-human growth fusion inpancreatic acinar cells of transgeneic mice. Nature 131: 600-603Palmiter, R. D., Behringer, R. R., Quaife, C. J., Maxwell, F., Maxwell,I. H., Brinster, R. L. (1987) Cell lineage ablation in transgeneic miceby cell-specific expression of a toxin gene. Cell 50: 435-443 T cellslck promoter Chaffin, K. E., Beals, C. R., Wilkie, T. M., Forbush, K.A., Simon, M. I., Perlmutter, R. M. (1990) EMBO Journal 9: 3821-3829 Bcells Immunoglobulin Borelli, E., Heyman, R., Hsi, M., Evans, R. M.(1988) Targeting of kappa light an inducible toxic phenotype in animalcells. Proc. Natl. Acad. chain Sci. USA 85: 7572-7576 Heyman, R. A.,Borrelli, E., Lesley, J., Anderson, D., Richmond, D. D., Baird, S. M.,Hyman, R., Evans, R. M. (1989) Thymidine kinase obliteration: creationof transgenic mice with controlled immunodeficiencies. Proc. Natl. Acad.Sci. USA 86: 2698-2702 Schwann cells P₀ promoter Messing, A., Behringer,R. R., Hammang, J. P. Palmiter, R D, Brinster, R L, Lemke, G., P0promoter directs espression of reporter and toxin genes to Schwann cellsof transgenic mice. Neuron 8: 507-520 1992 Myelin basic Miskimins, R.Knapp, L., Dewey, M J, Zhang, X. Cell and tissue- protein specificexpression of a heterologous gene under control of the myelin basicprotein gene promoter in trangenic mice. Brain Res Dev Brain Res 1992Vol 65: 217-21 spermatids protamine Breitman, M. L., Rombola, H.,Maxwell, I. H., Klintworth, G. K., Bernstein, A. (1990) Genetic ablationin transgenic mice with attenuated diphtheria toxin A gene. Mol. Cell.Biol. 10: 474-479 lung Lung surfacant Ornitz, D. M., Palmiter, R. D.,Hammer, R. E., Brinster, R. L., gene Swift, G. H., MacDonald, R. J.(1985) Specific expression of an elastase-human growth fusion inpancreatic acinar cells of transgeneic mice. Nature 131: 600-603adipocyte P2 Ross, S. R, Braves, R A, Spiegelman, B M Targetedexpression of a toxin gene to adipose tissue: transgenic mice resistantto obesity Genes and Dev 7: 1318-24 1993 muscle myosin light Lee, K J,Ross, R S, Rockman, H A, Harris, A N, O’Brien, T X, van- chain Bilsen,M., Shubeita, H E, Kandolf, R., Brem, G., Prices et al J. BIol. Chem.1992 Aug 5, 267: 15875-85 Alpha actin Muscat, G E., Perry, S., Prentice,H. Kedes, L. The human skeletal alpha-actin gene is regulated by amuscle-specific enhancer that binds three nuclear factors. GeneExpression 2, 111-26, 1992 neurons neurofilament Reeben, M. Halmekyto,M. Alhonen, L. Sinervirta, R. Saarma, M. Janne, J. proteinsTissue-specific expression of rat light neurofilament promoter-drivenreporter gene in transgenic mice. BBRC 1993: 192: 465-70 liver tyrosineaminotransferase, albumin, apolipoproteins

[0187] Target Gene Constructs

[0188] In embodiments of the invention in which the chimeric proteinsare designed such that their multimerization activates transcription ofa target gene, an appropriate target gene construct is also used in theengineered cells. Appropriate target gene constructs are thosecontaining a target gene and a cognate transcriptional control elementsuch as a promoter and/or enhancer which is responsive to themultimerization of the chimeric proteins. In embodiments involvingdirect activation of transcription, that responsiveness may be achievedby the presence in the target gene construct of one or more DNAsequences recognized by the DNA-binding domain of a chimeric protein ofthis invention (i.e., a DNA sequence to which the chimeric proteinbinds). In embodiments involving indirect activation of transcription,responsiveness may be achieved by the presence in the target geneconstruct of a promoter and/or enhancer sequence which is activated byan intracellular signal generated by multimerization of the chimericproteins. For example, where the chimeric proteins contain the TCR zetachain intracellular domain, the target gene is linked to and under theexpression control of the IL-2 promoter region.

[0189] This invention also provides target DNA constructs containing (a)a cognate DNA sequence, e.g. to which a DNA-binding chimeric protein ofthis invention is capable of binding (or which is susceptible toindirect activation as discussed above), and (b) flanking DNA sequencefrom the locus of a desired target gene endogenous to the host cells.These constructs permit homologous recombination of the cognate DNAsequence into a host cell in association with an endogenous target gene.In other embodiments the construct contains a desired gene and flankingDNA sequence from a target locus permitting the homologous recombinationof the target gene into the desired locus. Such a target construct mayalso contain the cognate DNA sequence, or the cognate DNA sequence maybe provided by the locus.

[0190] The target gene in any of the foregoing embodiments may encodefor example a surface membrane protein (such as a receptor protein), asecreted protein, a cytoplasmic protein, a nuclear protein, arecombinase such as Cre, a ribozyme or an antisense RNA. SeePCT/US94/01617 for general design and construction details and forvarious applications including gene therapy and see PCT/US95/10591regarding applications to animal models of disease.

[0191] This invention encompasses a variety of configurations for thechimeric proteins. In all cases involving the activation of target genetranscription, however, the chimeric proteins share an importantcharacteristic: cells containing constructs encoding the chimeras and atarget gene construct express the target gene at least one, preferablyat least two, and more preferably at least three or four or more ordersof magnitude more in the presence of the multimerizing ligand than inits absence. Optimally, expression of the selected gene is not observedunless the cells are or have been exposed to a multimerizing ligand.

[0192] To recap, the chimeric proteins are capable of initiating adetectable level of transcription of target genes within the engineeredcells upon exposure of the cells to the an improved rapalog, i.e.,following multimerization of the chimeras. Thus, transcription of targetgenes is activated in genetically engineered cells of this inventionfollowing exposure of the cells to an improved rapalog capable ofmultimerizing the chimeric protein molecules. Said differently,genetically engineered cells of this invention contain chimeric proteinsas described above and are responsive to the presence and/orconcentration of an improved rapalog which is capable of multimerizingthose chimeric protein molecules. That responsiveness is manifested bythe activation of transcription of a target gene. Such transcriptionalactivity can be readily detected by any conventional assays fortranscription of the target gene. In other embodiments, the biologicalresponse to ligand-mediated multimerization of the chimeras is celldeath or other biological events rather than direct activation oftranscription of a target gene.

[0193] Design and Assembly of the DNA Constructs

[0194] Constructs may be designed in accordance with the principles,illustrative examples and materials and methods disclosed in the patentdocuments and scientific literature cited herein, each of which isincorporated herein by reference, with modifications and furtherexemplification as described herein. Components of the constructs can beprepared in conventional ways, where the coding sequences and regulatoryregions may be isolated, as appropriate, ligated, cloned in anappropriate cloning host, analyzed by restriction or sequencing, orother convenient means. Particularly, using PCR, individual fragmentsincluding all or portions of a functional unit may be isolated, whereone or more mutations may be introduced using “primer repair”, ligation,in vitro mutagenesis, etc. as appropriate. In the case of DNA constructsencoding fusion proteins, DNA sequences encoding individual domains andsub-domains are joined such that they constitute a single open readingframe encoding a fusion protein capable of being translated in cells orcell lysates into a single polypeptide harboring all component domains.The DNA construct encoding the fusion protein may then be placed into avector that directs the expression of the protein in the appropriatecell type(s). For biochemical analysis of the encoded chimera, it may bedesirable to construct plasmids that direct the expression of theprotein in bacteria or in reticulocyte-lysate systems. For use in theproduction of proteins in mammalian cells, the protein-encoding sequenceis introduced into an expression vector that directs expression in thesecells. Expression vectors suitable for such uses are well known in theart. Various sorts of such vectors are commercially available.

[0195] Constructs encoding the chimeric proteins and target genes ofthis invention can be introduced into the cells as one or more DNAmolecules or constructs, in many cases in association with one or moremarkers to allow for selection of host cells which contain theconstruct(s). The construct(s) once completed and demonstrated to havethe appropriate sequences may then be introduced into a host cell by anyconvenient means. The constructs may be incorporated into vectorscapable of episomal replication (e.g. BPV or EBV vectors) or intovectors designed for integration into the host cells' chromosomes. Theconstructs may be integrated and packaged into non-replicating,defective viral genomes like Adenovirus, Adeno-associated virus (AAV),or Herpes simplex virus (HSV) or others, including retroviral vectors,for infection or transduction into cells. Viral delivery systems arediscussed in greater detail below. Alternatively, the construct may beintroduced by protoplast fusion, electro-poration, biolistics, calciumphosphate transfection, lipofection, microinjection of DNA or the like.The host cells will in some cases be grown and expanded in culturebefore introduction of the construct(s), followed by the appropriatetreatment for introduction of the construct(s) and integration of theconstruct(s). The cells will then be expanded and screened by virtue ofa marker present in the constructs. Various markers which may be usedsuccessfully include hprt, neomycin resistance, thymidine kinase,hygromycin resistance, etc., and various cell-surface markers such asTac, CD8, CD3, Thy1 and the NGF receptor.

[0196] In some instances, one may have a target site for homologousrecombination, where it is desired that a construct be integrated at aparticular locus. For example, one can delete and/or replace anendogenous gene (at the same locus or elsewhere) with a recombinanttarget construct of this invention. For homologous recombination, onemay generally use either Q or O-vectors. See, for example, Thomas andCapecchi, Cell (1987) 51, 503-512; Mansour, et al., Nature (1988) 336,348-352; and Joyner, et al., Nature (1989) 338, 153-156.

[0197] The constructs may be introduced as a single DNA moleculeencoding all of the genes, or different DNA molecules having one or moregenes. The constructs may be introduced simultaneously or consecutively,each with the same or different markers.

[0198] Vectors containing useful elements such as bacterial or yeastorigins of replication, selectable and/or amplifiable markers,promoter/enhancer elements for expression in procaryotes or eucaryotes,and mammalian expression control elements, etc. which may be used toprepare stocks of construct DNAs and for carrying out transfections arewell known in the art, and many are commercially available.

[0199] Delivery of Nuceic Acid: Ex Vivo and In Vivo

[0200] Any means for the introduction of heterologous nucleic acids intohost cells, especially eucaryotic cells, an in particular animal cells,preferably human or non-human mammalian cells, may be adapted to thepractice of this invention. For the purpose of this discussion, thevarious nucleic acid constructs described herein may together bereferred to as the transgene. Ex vivo approaches for delivery of DNAinclude calcium phosphate precipitation, electroporation, lipofectionand infection via viral vectors. Two general in vivo gene therapyapproaches include (a) the delivery of “naked”, lipid-complexed orliposome-formulated or otherwise formulated DNA and (b) the delivery ofthe heterologous nucleic acids via viral vectors. In the formerapproach, prior to formulation of DNA, e.g. with lipid, a plasmidcontaining a transgene bearing the desired DNA constructs may first beexperimentally optimized for expression (e.g., inclusion of an intron inthe 5′ untranslated region and elimination of unnecessary sequences(Felgner, et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA,e.g. with various lipid or liposome materials, may then be effectedusing known methods and materials and delivered to the recipient mammal.

[0201] While various viral vectors may be used in the practice of thisinvention, retroviral-, AAV- and adenovirus-based approaches are ofparticular interest. See, for example, Dubensky et al. (1984) Proc.Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science243,375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86,3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 andFerry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Thefollowing additional guidance on the choice and use of viral vectors maybe helpful to the practitioner.

Retroviral Vectors

[0202] Retroviruses are a class of RNA viruses in which the RNA genomeis reversely transcribed to DNA in the infected cell. The retroviralgenome can integrate into the host cell genome and requires three viralgenes, gag, pol and env, as well as the viral long terminal repeats(LTRs). The LTRs also act as enhancers and promoters for the viralgenes. The packaging sequence of the virus, (Ψ), allows the viral RNA tobe distinguished from other RNAs in the cell (Verma et al., Nature389:239-242, 1997). For expression of a foreign gene, the viral proteinsare replaced with the gene of interest in the viral vector, which isthen transfected into a packaging line containing the viral packagingcomponents. Packaged virus is secreted from the packaging line into theculture medium, which can then be used to infect cells in culture. Sinceretroviruses are unable to infect non-dividing cells, they have beenused primarily for ex vivo gene therapy.

AAV Vectors

[0203] Adeno-associated virus (AAV)-based vectors are of generalinterest as a delivery vehicle to various tissues, including muscle andlung. AAV vectors infect cells and stably integrate into the cellulargenome with high frequency. AAV can infect and integrate intogrowth-arrested cells (such as the pulmonary epithelium), and isnon-pathogenic.

[0204] The AAV-based expression vector to be used typically includes the145 nucleotide AAV inverted terminal repeats (ITRs) flanking arestriction site that can be used for subcloning of the transgene,either directly using the restriction site available, or by excision ofthe transgene with restriction enzymes followed by blunting of the ends,ligation of appropriate DNA linkers, restriction digestion, and ligationinto the site between the ITRs. The capacity of AAV vectors is about 4.4kb. The following proteins have been expressed using various AAV-basedvectors, and a variety of promoter/enhancers: neomycinphosphotransferase, chloramphenicol acetyl transferase, Fanconi's anemiagene, cystic fibrosis transmembrane conductance regulator, andgranulocyte macrophage colony-stimulating factor (Kotin, R. M., HumanGene Therapy 5:793-801, 1994, Table I). A transgene incorporating thevarious DNA constructs of this invention can similarly be included in anAAV-based vector. As an alternative to inclusion of a constitutivepromoter such as CMV to drive expression of the recombinant DNA encodingthe fusion protein(s), an AAV promoter can be used (ITR itself or AAV p5(Flotte, et al. J. Biol. Chem. 268:3781-3790, 1993)).

[0205] Such a vector can be packaged into AAV virions by reportedmethods. For example, a human cell line such as 293 can beco-transfected with the AAV-based expression vector and another plasmidcontaining open reading frames encoding AAV rep and cap under thecontrol of endogenous AAV promoters or a heterologous promoter. In theabsence of helper virus, the rep proteins Rep68 and Rep78 preventaccumulation of the replicative form, but upon superinfection withadenovirus or herpes virus, these proteins permit replication from theITRs (present only in the construct containing the transgene) andexpression of the viral capsid proteins. This system results inpackaging of the transgene DNA into AAV virions (Carter, B. J., CurrentOpinion in Biotechnology 3:533-539, 1992; Kotin, R. M, Human GeneTherapy 5:793-801, 1994)). Methods to improve the titer of AAV can alsobe used to express the transgene in an AAV virion. Such strategiesinclude, but are not limited to: stable expression of the ITR-flankedtransgene in a cell line followed by transfection with a second plasmidto direct viral packaging; use of a cell line that expresses AAVproteins inducibly, such as temperature-sensitive inducible expressionor pharmacologically inducible expression. Additionally, one mayincrease the efficiency of AAV transduction by treating the cells withan agent that facilitates the conversion of the single stranded form tothe double stranded form, as described in Wilson et al., WO96/39530.

[0206] Concentration and purification of the virus can be achieved byreported methods such as banding in cesium chloride gradients, as wasused for the initial report of AAV vector expression in vivo (Flotte, etal. J. Biol. Chem. 268:3781-3790, 1993) or chromatographic purification,as described in O'Riordan et al., WO97/08298.

[0207] For additional detailed guidance on AAV technology which may beuseful in the practice of the subject invention, including methods andmaterials for the incorporation of a transgene, the propagation andpurification of the recombinant AAV vector containing the transgene, andits use in transfecting cells and mammals, see e.g. Carter et al, U.S.Pat. No. 4,797,368 (10 Jan. 1989); Muzyczka et al, U.S. Pat. No.5,139,941 (18 Aug. 1992); Lebkowski et al, U.S. Pat. No. 5,173,414 (22Dec. 1992); Srivastava, U.S. Pat. No. 5,252,479 (12 Oct. 1993);Lebkowski et al, U.S. Pat. No. 5,354,678 (11 Oct. 1994); Shenk et al,U.S. Pat. No. 5,436,146 (25 Jul. 1995); Chatterjee et al, U.S. Pat. No.5,454,935 (12 Dec. 1995), Carter et al WO 93/24641 (published 9 Dec.1993), and Flotte et al., U.S. Pat. No. 5,658,776 (19 Aug. 1997).

Adenovirus Vectors

[0208] Various adenovirus vectors have been shown to be of use in thetransfer of genes to mammals, including humans. Replication-deficientadenovirus vectors have been used to express marker proteins and CFTR inthe pulmonary epithelium. The first generation E1a deleted adenovirusvectors have been improved upon with a second generation that includes atemperature-sensitive E2a viral protein, designed to express less viralprotein and thereby make the virally infected cell less of a target forthe immune system (Goldman et al., Human Gene Therapy 6:839-851, 1995).More recently, a viral vector deleted of all viral open reading frameshas been reported (Fisher et al., Virology 217:11-22, 1996). Moreover,it has been shown that expression of viral IL-10 inhibits the immuneresponse to adenoviral antigen (Qin et al., Human Gene Therapy8:1365-1374, 1997).

[0209] DNA sequences of a number of adenovirus types are available fromGenbank. The adenovirus DNA sequences may be obtained from any of the 41human adenovirus types currently identified. Various adenovirus strainsare available from the American Type Culture Collection, Rockville, Md.,or by request from a number of commercial and academic sources. Atransgene as described herein may be incorporated into any adenoviralvector and delivery protocol, by the same methods (restriction digest,linker ligation or filling in of ends, and ligation) used to insert theCFTR or other genes into the vectors. Hybrid Adenovirus-AAV vectorsrepresented by an adenovirus capsid containing selected portions of theadenovirus sequence, 5′ and 3′ AAV ITR sequences flanking the transgeneand other conventional vector regulatory elements may also be used. Seee.g. Wilson et al, International Patent Application Publication No. WO96/13598. For additional detailed guidance on adenovirus and hybridadenovirus-AAV technology which may be useful in the practice of thesubject invention, including methods and materials for the incorporationof a transgene, the propagation and purification of recombinant viruscontaining the transgene, and its use in transfecting cells and mammals,see also Wilson et al, WO 94/28938, WO 96/13597 and WO 96/26285, andreferences cited therein.

[0210] Generally the DNA or viral particles are transferred to abiologically compatible solution or pharmaceutically acceptable deliveryvehicle, such as sterile saline, or other aqueous or non-aqueousisotonic sterile injection solutions or suspensions, numerous examplesof which are well known in the art, including Ringer's, phosphatebuffered saline, or other similar vehicles.

[0211] Preferably, in gene therapy applications, the DNA or recombinantvirus is administered in sufficient amounts to transfect cells at alevel providing therapeutic benefit without undue adverse effects.Optimal dosages of DNA or virus depends on a variety of factors, asdiscussed elsewhere, and may thus vary somewhat from patient to patient.Again, therapeutically effective doses of viruses are considered to bein the range of about 20 to about 50 ml of saline solution containingconcentrations of from about 1×10⁷ to about 1×10¹⁰ pfu of virus/ml, e.g.from 1×10⁸ to 1×10⁹ pfu of virus/ml.

[0212] Host Cells

[0213] This invention is particularly useful for the engineering ofanimal cells and in applications involving the use of such engineeredanimal cells. The animal cells may be insect, worm or mammalian cells.While various mammalian cells may be used, including, by way of example,equine, bovine, ovine, canine, feline, murine, and non-human primatecells, human cells are of particular interest. Among the variousspecies, various types of cells may be used, such as hematopoietic,neural, glial, mesenchymal, cutaneous, mucosal, stromal, muscle(including smooth muscle cells), spleen, reticulo-endothelial,epithelial, endothelial, hepatic, kidney, gastrointestinal, pulmonary,fibroblast, and other cell types. Of particular interest arehematopoietic cells, which may include any of the nucleated cells whichmay be involved with the erythroid, lymphoid or myelomonocytic lineages,as well as myoblasts and fibroblasts. Also of interest are stem andprogenitor cells, such as hematopoietic, neural, stromal, muscle,hepatic, pulmonary, gastrointestinal and mesenchymal stem cells

[0214] The cells may be autologous cells, syngeneic cells, allogeneiccells and even in some cases, xenogeneic cells with respect to anintended host organism. The cells may be modified by changing the majorhistocompatibility complex (“MHC”) profile, by inactivatingβ2-microglobulin to prevent the formation of functional Class I MHCmolecules, inactivation of Class II molecules, providing for expressionof one or more MHC molecules, enhancing or inactivating cytotoxiccapabilities by enhancing or inhibiting the expression of genesassociated with the cytotoxic activity, or the like.

[0215] In some instances specific clones or oligoclonal cells may be ofinterest, where the cells have a particular specificity, such as T cellsand B cells having a specific antigen specificity or homing target sitespecificity.

[0216] Introduction of Constructs into Animals

[0217] Cells which have been modified ex vivo with the DNA constructsmay be grown in culture under selective conditions and cells which areselected as having the desired construct(s) may then be expanded andfurther analyzed, using, for example, the polymerase chain reaction fordetermining the presence of the construct in the host cells and/orassays for the production of the desired gene product(s). Once modifiedhost cells have been identified, they may then be used as planned, e.g.grown in culture or introduced into a host organism.

[0218] Depending upon the nature of the cells, the cells may beintroduced into a host organism, e.g. a mammal, in a wide variety ofways. Hematopoietic cells may be administered by injection into thevascular system, there being usually at least about 10⁴ cells andgenerally not more than about 10¹⁰ cells. The number of cells which areemployed will depend upon a number of circumstances, the purpose for theintroduction, the lifetime of the cells, the protocol to be used, forexample, the number of administrations, the ability of the cells tomultiply, the stability of the therapeutic agent, the physiologic needfor the therapeutic agent, and the like. Generally, for myoblasts orfibroblasts for example, the number of cells will be at least about 10⁴and not more than about 10⁹ and may be applied as a dispersion,generally being injected at or near the site of interest. The cellswill, usually be in a physiologically-acceptable medium.

[0219] Cells engineered in accordance with this invention may also beencapsulated, e.g. using conventional biocompatible materials andmethods, prior to implantation into the host organism or patient for theproduction of a therapeutic protein. See e.g. Hguyen et al, TissueImplant Systems and Methods for Sustaining viable High Cell Densitieswithin a Host, U.S. Pat. No. 5,314,471 (Baxter International, Inc.);Uludag and Sefton, 1993, J. Biomed. Mater. Res. 27(10):1213-24 (HepG2cells/hydroxyethyl methacrylate-methyl methacrylate membranes); Chang etal, 1993, Hum Gene Ther 4(4):433-40 (mouse Ltk-cells expressinghGH/immunoprotective perm-selective alginate microcapsules; Reddy et al,1993, J Infect Dis 168(4):1082-3 (alginate); Tai and Sun, 1993, FASEB J7(11):1061-9 (mouse fibroblasts expressinghGH/alginate-poly-L-lysine-alginate membrane); Ao et al, 1995,Transplanataion Proc. 27(6):3349, 3350 (alginate); Rajotte et al, 1995,Transplantation Proc. 27(6):3389 (alginate); Lakey et al, 1995,Transplantation Proc. 27(6):3266 (alginate); Korbutt et al, 1995,Transplantation Proc. 27(6):3212 (alginate); Dorian et al, U.S. Pat. No.5,429,821 (alginate); Emerich et al, 1993, Exp Neurol 122(1):37-47(polymer-encapsulated PC12 cells); Sagen et al, 1993, J Neurosci13(6):2415-23 (bovine chromaffin cells encapsulated in semipermeablepolymer membrane and implanted into rat spinal subarachnoid space);Aebischer et al, 1994, Exp Neurol 126(2):151-8 (polymer-encapsulated ratPC12 cells implanted into monkeys; see also Aebischer, WO 92/19595);Savelkoul et al, 1994, J Immunol Methods 170(2):185-96 (encapsulatedhybridomas producing antibodies; encapsulated transfected cell linesexpressing various cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8(engineered BHK cells expressing human nerve growth factor encapsulatedin an immunoisolation polymeric device and transplanted into rats);Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats);Kordower et al, 1994, PNAS USA 91(23):10898-902 (polymer-encapsulatedengineered BHK cells expressing hNGF implanted into monkeys) and Butleret al WO 95/04521 (encapsulated device). The cells may then beintroduced in encapsulated form into an animal host, preferably a mammaland more preferably a human subject in need thereof. Preferably theencapsulating material is semipermeable, permitting release into thehost of secreted proteins produced by the encapsulated cells. In manyembodiments the semipermeable encapsulation renders the encapsulatedcells immunologically isolated from the host organism in which theencapsulated cells are introduced. In those embodiments the cells to beencapsulated may express one or more chimeric proteins containingcomponent domains derived from proteins of the host species and/or fromviral proteins or proteins from species other than the host species. Forexample in such cases the chimeras may contain elements derived fromGAL4 and VP16. The cells may be derived from one or more individualsother than the recipient and may be derived from a species other thanthat of the recipient organism or patient.

[0220] Instead of ex vivo modification of the cells, in many situationsone may wish to modify cells in vivo. For this purpose, varioustechniques have been developed for modification of target tissue andcells in vivo. A number of viral vectors have been developed, such asadenovirus, adeno-associated virus, and retroviruses, as discussedabove, which allow for transfection and, in some cases, integration ofthe virus into the host. See, for example, Dubensky et al. (1984) Proc.Natl. Acad. Sci. USA 81, 7529-7533; Kaneda et al., (1989) Science243,375-378; Hiebert et al. (1989) Proc. Natl. Acad. Sci. USA 86,3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265, 17285-17293 andFerry, et al. (1991) Proc. Natl. Acad. Sci. USA 88, 8377-8381. Thevector may be administered by injection, e.g. intravascularly orintramuscularly, inhalation, or other parenteral mode. Non-viraldelivery methods such as administration of the DNA via complexes withliposomes or by injection, catheter or biolistics may also be used.

[0221] In accordance with in vivo genetic modification, the manner ofthe modification will depend on the nature of the tissue, the efficiencyof cellular modification required, the number of opportunities to modifythe particular cells, the accessibility of the tissue to the DNAcomposition to be introduced, and the like. By employing an attenuatedor modified retrovirus carrying a target transcriptional initiationregion, if desired, one can activate the virus using one of the subjecttranscription factor constructs, so that the virus may be produced andtransfect adjacent cells.

[0222] The DNA introduction need not result in integration in everycase. In some situations, transient maintenance of the DNA introducedmay be sufficient. In this way, one could have a short term effect,where cells could be introduced into the host and then turned on after apredetermined time, for example, after the cells have been able to hometo a particular site.

[0223] Binding properties, Assays

[0224] Rapamycin is known to bind to the human protein, FKBP12 and toform a tripartite complex with hFKBP12 and FRAP, a human counterpart tothe yeast proteins TOR1 and TOR2. Rapalogs may be characterized andcompared to rapamycin with respect to their ability to bind to humanFKBP12 and/or to form tripartite complexes with human FKBP12 and humanFRAP (or fusion proteins or fragments containing its FRB domain). See WO96/41865 (Clackson et al). That application discloses various materialsand methods which can be used to quantify the ability of a compound tobind to human FKBP12 or to form a tripartite complex with (i.e.,“heterodimerize”) proteins comprising human FKBP12 and the FRB domain ofhuman FRAP, respectively. Such assays include fluorescence polarizationassays to measure binding. Also included are cell based transcriptionassays in which the ability of a compound to form the tripartite complexis measured indirectly by correlation with the observed level ofreporter gene product produced by engineered mammalian cells in thepresence of the compound. Corresponding cell-based assays may also beconducted in engineered yeast cells. See e.g. WO 95/33052 (Berlin etal).

[0225] It will often be preferred that the rapalogs of this invention bephysiologically acceptable (i.e., lack undue toxicity toward the cell ororganism with which it is to be used), can be taken orally by animals(i.e., is orally active in applications in whole animals, including genetherapy), and/or can cross cellular and other membranes, as necessaryfor a particular application.

[0226] In addition, preferred rapalogs are those which bindpreferentially to mutant immunophilins (by way of non-limiting example,a human FKBP in which Phe36 is replaced with a different amino acid,preferably an amino acid with a less bulky R group such as valine oralanine) over native or naturally-ocurring immunophilins. For example,such compounds may bind preferentially to mutant FKBPs at least an orderof magnitude better than they bind to human FKBP12, and in some casesmay bind to mutant FKBPs greater than 2 or even 3 or more orders ofmagnitude better than they do to human FKBP12, as determined by anyscientifically valid or art-accepted assay methodology.

[0227] Binding affinities of various rapalogs of this invention withrespect to human FKBP12, variants thereof or other immunophilin proteinsmay be determined by adaptation of known methods used in the case ofFKBP. For instance, the practitioner may measure the ability of acompound of this invention to compete with the binding of a known ligandto the protein of interest. See e.g. Sierkierka et al, 1989, Nature 341,755-757 (test compound competes with binding of labeled FK506 derivativeto FKBP).

[0228] One set of preferred rapalogs of this invention which binds, tohuman FKBP12, to a mutant thereof as discussed above, or to a fusionprotein containing such FKBP domains, with a Kd value below about 200nM, more preferably below about 50 nM, even more preferably below about10 nM, and even more preferably below about 1 nM, as measured by directbinding measurement (e.g. fluorescence quenching), competition bindingmeasurement (e.g. versus FK506), inhibition of FKBP enzyme activity(rotamase), or other assay methodology. In one subset of such compounds,the FKBP domain is one in which phenylalanine at position 36 has beenreplaced with an amino acid having a less bulky side chain, e.g.alanine, valine, methionine or serine.

[0229] A Competitive Binding FP Assay is described in detail in theexamples which follow. That assay permits the in vitro measurement of anIC50 value for a given compound which reflects its ability to bind to anFKBP protein in competition with a labeled FKBP ligand, such as, forexample, FK506.

[0230] One preferred class of compounds of this invention are thoserapalogs which have an IC50 value in the Competitive Binding FP Assaybetter than 1000 nM, preferably better than 300 nM, more preferablybetter than 100 nM, and even more preferably better than 10 nM withrespect to a given FKBP domain and ligand pair, e.g. human FKBP12 or avariant thereof with up to 10, preferably up to 5 amino acidreplacements, with a flouresceinated FK506 standard. In one subset ofthat class, the FKBP domain has one of the abovementioned modificationsat position 36.

[0231] The ability of the rapalogs to multimerize chimeric proteins maybe measured in cell-based assays by measuring the occurrence of an eventtriggered by such multimerization. For instance, one may use cellscontaining and capable of expressing DNA encoding a first chimericprotein comprising one or more FKBP-domains and one or more effectordomains as well as DNA encoding a second chimeric protein containing anFRB domain and one or more effector domains capable, uponmultimerization, of actuating a biological response. We prefer to usecells which further contain a reporter gene under the transcriptionalcontrol of a regulatory element (i.e., promoter) which is responsive tothe multimerization of the chimeric proteins. The design and preparationof illustrative components and their use in so engineered cells isdescribed in WO96/41865 and the other international patent applicationsreferred to in this and the foregoing section. The cells are grown ormaintained in culture. A rapalog is added to the culture medium andafter a suitable incubation period (to permit gene expression andsecretion, e.g. several hours or overnight) the presence of the reportergene product is measured. Positive results, i.e., multimerization,correlates with transcription of the reporter gene as observed by theappearance of the reporter gene product. The reporter gene product maybe a conveniently detectable protein (e.g. by ELISA) or may catalyze theproduction of a conveniently detectable product (e.g. colored).Materials and methods for producing appropriate cell lines forconducting such assays are disclosed in the international patentapplications cited above in this section. Typically used target genesinclude by way of example SEAP, hGH, beta-galactosidase, GreenFluorescent Protein and luciferase, for which convenient assays arecommercially available.

[0232] Another preferred class of compounds of this invention are thosewhich are capable of inducing a detectable signal in a 2-hybridtranscription assay based on fusion proteins containing an FKBP domain.Preferably, the FKBP domain is an FKBP domain other than wild-type humanFKBP12.

[0233] Another assay for measuring the ability of the rapalogs tomultimerize chimeric proteins, like the FKBP-based transcription assay,is a cell-based assay which measures the occurrence of an eventtriggered by such multimerization. In this case, one uses cells whichconstitutively express a detectable product. The cells also contain andare capable of expressing DNAs encoding chimeric proteins comprising oneor more immunophilin-derived ligand binding domains and one or moreeffector domains, such as the intracellular domain of FAS, capable, uponmultimerization, of triggering cell death. The design and preparation ofillustrative components and their use in so engineering cells isdescribed in WO95/02684. See also WO96/41865. The cells are maintaininedor cultured in a culture medium permitting cell growth or continuedviability. The cells or medium are assayed for the presence of theconstitutive cellular product, and a base-line level of reporter is thusestablished. One may use cells engineered for constitutive production ofhGH or any other conveniently detectable product to serve as thereporter. The compound to be tested is addded to the medium, the cellsare incubated, and the cell lysate or medium is tested for the presenceof reporter at one or more time points. Decrease in reporter productionindicates cell death, an indirect measure of multimerization of thefusion proteins.

[0234] Another preferred class of compounds of this invention are thosewhich are capable of inducing a detectable signal in such anFKBP/FRB-based apoptosis assay. Preferably, the FKBP domain is an FKBPdomain other than wild-type human FKBP12. In some cases, the FKBP domainis modified, as discussed above. Also preferably, the FRB domain is anFRB domain other than wild-type FRB from human FRAP. In some cases, theFRB domain is modified at position 2098, as described above.

[0235] Conducting such assays permits the practitioner to selectrapalogs possessing the desired IC50 values and/or binding preferencefor a mutant FKBP over wild-type human FKBP12. The Competitive BindingFP Assay permits one to select monomers or rapalogs which possess thedesired IC50 values and/or binding preference for a mutant FKBP orwild-type FKBP relative to a control, such as FK506.

[0236] Applications

[0237] The rapalogs can be used as described in WO94/18317, WO95/02684,WO96/20951, WO95/41865, e.g. to regulatably activate the transcriptionof a desired gene, delete a target gene, actuate apoptosis, or triggerother biological events in engineered cells growing in culture or inwhole organisms, including in gene therapy applications. The followingare non-limiting examples of applications of the subject invention.

[0238] 1. Regulated gene therapy. In many instances, the ability toswitch a therapeutic gene on and off at will or the ability to titrateexpression with precision are important for therapeutic efficacy. Thisinvention is particularly well suited for achieving regulated expressionof a therapeutic target gene in the context of human gene therapy. Oneexample uses a pair of chimeric proteins (one containing at least oneFRB domain, the other containing at least one FKBP domain), an improvedrapalog of this invention capable of dimerizing the chimeras, and atarget gene construct to be expressed. One of the chimeric proteinscomprises a DNA-binding domain, preferably a composite DNA-bindingdomain as described in Pomerantz et al, supra, as the heterologouseffector domain. The second chimeric protein comprises a transcriptionalactivating domain as the heterologous effector domain. The improvedrapalog is capable of binding to both chimeras and thus of effectivelycross-linking the chimeras. DNA molecules encoding and capable ofdirecting the expression of these chimeric proteins are introduced intothe cells to be engineered. Also introduced into the cells is a targetgene linked to a DNA sequence to which the DNA-binding domain is capableof binding. Contacting the engineered cells or their progeny with theimproved rapalog (by administering it to the animal or patient) leads toassembly of the transcription factor complex and hence to expression ofthe target gene. The design and use of similar components is disclosedin PCT/US93/01617 and in WO 96/41865 (Clackson et al). In practice, thelevel of target gene expression should be a function of the number orconcentration of chimeric transcription factor complexes, which shouldin turn be a function of the concentration of the improved rapalog. Dose(of improved rapalog)-responsive gene expression is typically observed.

[0239] The improved rapalog may be administered to the patient asdesired to activate transcription of the target gene. Depending upon thebinding affinity of the improved rapalog, the response desired, themanner of administration, the biological half-life of the rapalog and/ortarget gene mRNA, the number of engineered cells present, variousprotocols may be employed. The improved rapalog may be administered byvarious routes, including parenterally or orally. The number ofadministrations will depend upon the factors described above. Theimproved rapalog may be taken orally as a pill, powder, or dispersion;bucally; sublingually; injected intravascularly, intraperitoneally,intramuscularly, subcutaneously; by inhalation, or the like. Theimproved rapalog (and monomeric antagonist compound) may be formulatedusing conventional methods and materials well known in the art for thevarious routes of administration. The precise dose and particular methodof administration will depend upon the above factors and be determinedby the attending physician or human or animal healthcare provider. Forthe most part, the manner of administration will be determinedempirically.

[0240] In the event that transcriptional activation by the improvedrapalog is to be reversed or terminated, a monomeric compound which cancompete with the improved rapalog may be administered. Thus, in the caseof an adverse reaction or the desire to terminate the therapeuticeffect, an antagonist to the dimerizing agent can be administered in anyconvenient way, particularly intravascularly, if a rapid reversal isdesired. Alternatively, one may provide for the presence of aninactivation domain (or transcriptional silencer) with a ligand bindingdomain. In another approach, cells may be eliminated through apoptosisvia signalling through Fas or TNF receptor as described elsewhere. SeeInternational Patent Applications PCT/US94/01617 and PCT/US94/08008.

[0241] The particular dosage of the improved rapalog for any applicationmay be determined in accordance with the procedures used for therapeuticdosage monitoring, where maintenance of a particular level of expressionis desired over an extended period of times, for example, greater thanabout two weeks, or where there is repetitive therapy, with individualor repeated doses of improved rapalog over short periods of time, withextended intervals, for example, two weeks or more. A dose of theimproved rapalog within a predetermined range would be given andmonitored for response, so as to obtain a time-expression levelrelationship, as well as observing therapeutic response. Depending onthe levels observed during the time period and the therapeutic response,one could provide a larger or smaller dose the next time, following theresponse. This process would be iteratively repeated until one obtaineda dosage within the therapeutic range. Where the improved rapalog ischronically administered, once the maintenance dosage of the improvedrapalog is determined, one could then do assays at extended intervals tobe assured that the cellular system is providing the appropriateresponse and level of the expression product.

[0242] It should be appreciated that the system is subject to manyvariables, such as the cellular response to the improved rapalog, theefficiency of expression and, as appropriate, the level of secretion,the activity of the expression product, the particular need of thepatient, which may vary with time and circumstances, the rate of loss ofthe cellular activity as a result of loss of cells or expressionactivity of individual cells, and the like.

[0243] 2. Production of recombinant proteins and viruses. Production ofrecombinant therapeutic proteins for commercial and investigationalpurposes is often achieved through the use of mammalian cell linesengineered to express the protein at high level. The use of mammaliancells, rather than bacteria or yeast, is indicated where the properfunction of the protein requires post-translational modifications notgenerally performed by heterologous cells. Examples of proteins producedcommercially this way include erythropoietin, tissue plasminogenactivator, clotting factors such as Factor VIII:c, antibodies, etc. Thecost of producing proteins in this fashion is directly related to thelevel of expression achieved in the engineered cells. A secondlimitation on the production of such proteins is toxicity to the hostcell: Protein expression may prevent cells from growing to high density,sharply reducing production levels. Therefore, the ability to tightlycontrol protein expression, as described for regulated gene therapy,permits cells to be grown to high density in the absence of proteinproduction. Only after an optimum cell density is reached, is expressionof the gene activated and the protein product subsequently harvested.

[0244] A similar problem is encountered in the construction and use of“packaging lines” for the production of recombinant viruses forcommercial (e.g., gene therapy) and experimental use. These cell linesare engineered to produce viral proteins required for the assembly ofinfectious viral particles harboring defective recombinant genomes.Viral vectors that are dependent on such packaging lines includeretrovirus, adenovirus, and adeno-associated virus. In the latter case,the titer of the virus stock obtained from a packaging line is directlyrelated to the level of production of the viral rep and core proteins.But these proteins are highly toxic to the host cells. Therefore, it hasproven difficult to generate high-titer recombinant AAV viruses. Thisinvention provides a solution to this problem, by allowing theconstruction of packaging lines in which the rep and core genes areplaced under the control of regulatable transcription factors of thedesign described here. The packaging cell line can be grown to highdensity, infected with helper virus, and transfected with therecombinant viral genome. Then, expression of the viral proteins encodedby the packaging cells is induced by the addition of dimerizing agent toallow the production of virus at high titer.

[0245] 3. Biological research. This invention is applicable to a widerange of biological experiments in which precise control over a targetgene is desired. These include: (1) expression of a protein or RNA ofinterest for biochemical purification; (2) regulated expression of aprotein or RNA of interest in tissue culture cells (or in vivo, viaengineered cells) for the purposes of evaluating its biologicalfunction; (3) regulated expression of a protein or RNA of interest intransgenic animals for the purposes of evaluating its biologicalfunction; (4) regulating the expression of a gene encoding anotherregulatory protein, ribozyme or antisense molecule that acts on anendogenous gene for the purposes of evaluating the biological functionof that gene. Transgenic animal models and other applications in whichthe components of this invention may be adapted include those disclosedin PCT/US95/10591.

[0246] This invention further provides kits useful for the foregoingapplications. Such kits contain DNA constructs encoding and capable ofdirecting the expression of chimeric proteins of this invention (and maycontain additional domains as discussed above) and, in embodimentsinvolving regulated gene transcription, a target gene constructcontaining a target gene linked to one or more transcriptioal controlelements which are activated by the multimerization of the chimericproteins. Alternatively, the target gene construct may contain a cloningsite for insertion of a desired target gene by the practitioner. Suchkits may also contain a sample of a dimerizing agent capable ofdimerizing the two recombinant proteins and activating transcription ofthe target gene.

[0247] Formulations, Dosage and Administration

[0248] By virtue of its capacity to promote protein-proteininteractions, a rapalog of this invention may be used in pharmaceuticalcompositions and methods for promoting formation of complexes ofchimeric proteins of this invention in a human or non-human mammalcontaining genetically engineered cells of this invention.

[0249] The preferred method of such treatment or prevention is byadministering to the mammal an effective amount of the compound topromote measurable formation of such complexes in the engineered cells,or preferably, to promote measurable actuation of the desired biologicalevent triggered by such complexation, e.g. transcription of a targetgene, apoptosis of engineered cells, etc.

[0250] Therapeutic/Prophylactic Administration & PharmaceuticalCompositions

[0251] The rapalogs can exist in free form or, where appropriate, insalt form. Pharmaceutically acceptable salts of many types of compoundsand their preparation are well-known to those of skill in the art. Thepharmaceutically acceptable salts of compounds of this invention includethe conventional non-toxic salts or the quaternary ammonium salts ofsuch compounds which are formed, for example, from inorganic or organicacids of bases.

[0252] The compounds of the invention may form hydrates or solvates. Itis known to those of skill in the art that charged compounds formhydrated species when lyophilized with water, or form solvated specieswhen concentrated in a solution with an appropriate organic solvent.

[0253] This invention also relates to pharmaceutical compositionscomprising a therapeutically (or prophylactically) effective amount ofthe compound, and one or more pharmaceutically acceptable carriersand/or other excipients. Carriers include e.g. saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof, and arediscussed in greater detail below. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. The composition can be a liquid solution, suspension, emulsion,tablet, pill, capsule, sustained release formulation, or powder. Thecomposition can be formulated as a suppository, with traditional bindersand carriers such as triglycerides. Oral formulation can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc. Formulation may involve mixing, granulating andcompressing or dissolving the ingredients as appropriate to the desiredpreparation.

[0254] The pharmaceutical carrier employed may be, for example, either asolid or liquid.

[0255] Illustrative solid carrier include lactose, terra alba, sucrose,talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acidand the like. A solid carrier can include one or more substances whichmay also act as flavoring agents, lubricants, solubilizers, suspendingagents, fillers, glidants, compression aids, binders ortablet-disintegrating agents; it can also be an encapsulating material.In powders, the carrier is a finely divided solid which is in admixturewith the finely divided active ingredient. In tablets, the activeingredient is mixed with a carrier having the necessary compressionproperties in suitable proportions, and compacted in the shape and sizedesired. The powders and tablets preferably contain up to 99% of theactive ingredient. Suitable solid carriers include, for example, calciumphosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch,gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose,polyvinylpyrrolidine, low melting waxes and ion exchange resins.

[0256] Illustrative liquid carriers include syrup, peanut oil, oliveoil, water, etc. Liquid carriers are used in preparing solutions,suspensions, emulsions, syrups, elixirs and pressurized compositions.The active ingredient can be dissolved or suspended in apharmaceutically acceptable liquid carrier such as water, an organicsolvent, a mixture of both or pharmaceutically acceptable oils or fats.The liquid carrier can contain other suitable pharmaceutical additivessuch as solubilizers, emulsifiers, buffers, preservatives, sweeteners,flavoring agents, suspending agents, thickening agents, colors,viscosity regulators, stabilizers or osmo-regulators. Suitable examplesof liquid carriers for oral and parenteral administration include water(partially containing additives as above, e.g. cellulose derivatives,preferably sodium carboxymethyl cellulose solution), alcohols (includingmonohydric alcohols and polyhydric alcohols, e.g. glycols) and theirderivatives, and oils (e.g. fractionated coconut oil and arachis oil).For parenteral administration, the carrier can also be an oily estersuch as ethyl oleate and isopropyl myristate. Sterile liquid carriersare useful in sterile liquid form compositions for parenteraladministration. The liquid carrier for pressurized compositions can behalogenated hydrocarbon or other pharmaceutically acceptable propellant.Liquid pharmaceutical compositions which are sterile solutions orsuspensions can be utilized by, for example, intramuscular,intraperitoneal or subcutaneous injection. Sterile solutions can also beadministered intravenously. The compound can also be administered orallyeither in liquid or solid composition form.

[0257] The carrier or excipient may include time delay material wellknown to the art, such as glyceryl monostearate or glyceryl distearatealong or with a wax, ethylcellulose, hydroxypropylmethylcellulose,methylmethacrylate and the like. When formulated for oraladministration, 0.01% Tween 80 in PHOSAL PG-50 (phospholipid concentratewith 1,2-propylene glycol, A. Nattermann & Cie. GmbH) has beenrecognized as providing an acceptable oral formulation for othercompounds, and may be adapted to formulations for various compounds ofthis invention.

[0258] A wide variety of pharmaceutical forms can be employed. If asolid carrier is used, the preparation can be tableted, placed in a hardgelatin capsule in powder or pellet form or in the form of a troche orlozenge. The amount of solid carrier will vary widely but preferablywill be from about 25 mg to about 1 g. If a liquid carrier is used, thepreparation will be in the form of a syrup, emulsion, soft gelatincapsule, sterile injectable solution or suspension in an ampule or vialor nonaqueous liquid suspension.

[0259] To obtain a stable water soluble dosage form, a pharmaceuticallyacceptable salt of the multimerizer may be dissolved in an aqueoussolution of an organic or inorganic acid, such as a 0.3M solution ofsuccinic acid or citric acid. Alternatively, acidic derivatives can bedissolved in suitable basic solutions. If a soluble salt form is notavailable, the compound is dissolved in a suitable cosolvent orcombinations thereof. Examples of such suitable cosolvents include, butare not limited to, alcohol, propylene glycol, polyethylene glycol 300,polysorbate 80, glycerin, polyoxyethylated fatty acids, fatty alcoholsor glycerin hydroxy fatty acids esters and the like in concentrationsranging from 0-60% of the total volume.

[0260] Various delivery systems are known and can be used to administerthe multimerizer, or the various formulations thereof, includingtablets, capsules, injectable solutions, encapsulation in liposomes,microparticles, microcapsules, etc. Methods of introduction include butare not limited to dermal, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, pulmonary, epidural, ocular and(as is usually preferred) oral routes. The compound may be administeredby any convenient or otherwise appropriate route, for example byinfusion or bolus injection, by absorption through epithelial ormucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa,etc.) and may be administered together with other biologically activeagents. Administration can be systemic or local. For treatment orprophylaxis of nasal, bronchial or pulmonary conditions, preferredroutes of administration are oral, nasal or via a bronchial aerosol ornebulizer.

[0261] In certain embodiments, it may be desirable to administer thecompound locally to an area in need of treatment; this may be achievedby, for example, and not by way of limitation, local infusion duringsurgery, topical application, by injection, by means of a catheter, bymeans of a suppository, or by means of a skin patch or implant, saidimplant being of a porous, non-porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

[0262] In a specific embodiment, the composition is formulated inaccordance with routine procedures as a pharmaceutical compositionadapted for intravenous administration to human beings. Typically,compositions for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic to ease pain at theside of the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as alyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

[0263] Administration to an individual of an effective amount of thecompound can also be accomplished topically by administering thecompound(s) directly to the affected area of the skin of the individual.For this purpose, the compound is administered or applied in acomposition including a pharmacologically acceptable topical carrier,such as a gel, an ointment, a lotion, or a cream, which includes,without limitation, such carriers as water, glycerol, alcohol, propyleneglycol, fatty alcohols, triglycerides, fatty acid esters, or mineraloils.

[0264] Other topical carriers include liquid petroleum, isopropylpalmitate, polyethylene glycol, ethanol (95%), polyoxyethylenemonolaurate (5%) in water, or sodium lauryl sulfate (5%) in water. Othermaterials such as anti-oxidants, humectants, viscosity stabilizers, andsimilar agents may be added as necessary. Percutaneous penetrationenhancers such as Azone may also be included.

[0265] In addition, in certain instances, it is expected that thecompound may be disposed within devices placed upon, in, or under theskin. Such devices include patches, implants, and injections whichrelease the compound into the skin, by either passive or active releasemechanisms.

[0266] Materials and methods for producing the various formulations arewell known in the art and may be adapted for practicing the subjectinvention. See e.g. U.S. Pat. Nos. 5,182,293 and 4,837,311 (tablets,capsules and other oral formulations as well as intravenousformulations) and European Patent Application Publication Nos. 0 649 659(published Apr. 26, 1995; illustrative formulation for IVadministration) and 0 648 494 (published Apr. 19, 1995; illustrativeformulation for oral administration).

[0267] The effective dose of the compound will typically be in the rangeof about 0.01 to about 50 mg/kgs, preferably about 0.1 to about 10 mg/kgof mammalian body weight, administered in single or multiple doses.Generally, the compound may be administered to patients in need of suchtreatment in a daily dose range of about 1 to about 2000 mg per patient.

[0268] The amount of compound which will be effective in the treatmentor prevention of a particular disorder or condition will depend in parton the characteristics of the fusion proteins to be multimerized, thecharacteristics and location of the genetically engineered cells, and onthe nature of the disorder or condition, which can be determined bystandard clinical techniques. In addition, in vitro or in vivo assaysmay optionally be employed to help identify optimal dosage ranges.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems. The precise dosage levelshould be determined by the attending physician or other health careprovider and will depend upon well known factors, including route ofadministration, and the age, body weight, sex and general health of theindividual; the nature, severity and clinical stage of the disease; theuse (or not) of concomitant therapies; and the nature and extent ofgenetic engineering of cells in the patient.

[0269] The invention also provides a pharmaceutical pack or kitcomprising one or more containers containing one or more of theingredients of the pharmaceutical compositions of the invention.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceutical or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. The notice or package insert may contain instructionsfor use of an improved rapalog of this invention, consistent with thedisclsoure herein.

EXAMPLES Example 1 Synthesis of Representative C-24 modified Rapalogs

[0270] 1.1. Rapamycin purification. Rapamycin was obtained byfermentation. The rapamycin producing organism, Streptomyceshygroscopicus (ATCC# 29253), was cultivated on a complex media in 15 Lor 30 L fed-batch fermentations. The biomass was harvested after 9-14days by centrifugation. The supernatant was contacted for 1-2 hours witha nonionic, polymeric adsorbent resin, XAD-16 (Rohm and Haas). Theadsorbent was recovered by centrifugation, combined with the biomass,and extracted repeatedly with methylene chloride. The solvent wasremoved in vacuo and the resulting residue extracted with acetonitrilewhich was then condensed in a similar manner. Chromatographicpurification of the crude rapamycin was achieved by flash chromatographyon silica gel (40% Acetone/Hexanes) followed by C-18 reversed-phase HPLC(70% CH3CN/H2O). Rapamycin obtained exhibited identical HPLC,spectroscopic, and biological characteristics as an authentic sample ofrapamycin.

[0271] 1.2. Rapamycin (E and Z)-24-(O-methyloxime) (5, 6)(GeneralProcedure)

[0272] A solution of rapamycin (60 mg 65.6 mmol) in MeOH (2 mL) wastreated with NaOAc (22 mg 262 mmol, 4.0 eq) followed by methoxy]aminehydrochloride (22 mg 262 mmol, 4.0 eq) and stirred at room temperaturefor 48 h. After this time the reaction mixture was quenched with H2O (10mL) and extracted with EtOAc (3×10 ml). The combined organic extractswere washed with saturated NaCl solution (2×10 mL), dried over Na2 SO4,filtered, and the solution concentrated in vacuo. The resulting residuewas subjected to flash chromatography on silica gel (10%MeOH/dichloromethane) to afford a mixture of isomers. The isomer mixturewas separated by HPLC (35%

25% H2O/MeCN through a Kromasil C-18 250×20 mm column, 12 mL/min) toprovide 13 mg (21%) of the faster eluting Z isomer and 7.6 mg (12%) ofthe E isomer. Z isomer: high-resolution mass spectrum (FAB) m/z 965.5749[(M+Na)+, calcd for C52H82N2O13Na 965.5710]. E isomer: high-resolutionmass spectrum (FAB) m/z 965.5701 [(M+Na)+, calcd for C52 H82N2O13Na965.5710].

[0273] 1.3. Rapamycin (E and Z)-24-(O-ethyloxime) (7, 8)

[0274] Prepared in an analogous manner to Rapamycin (E andZ)-24-(O-methyloxime). The isomer mixture was separated by HPLC (30%H2O/MeCN through a Kromasil C-18 250×20 mm column, 12 mL/min) to provide7.7 mg (25%) of the faster eluting Z isomer and 0.5 mg (2%) of the Eisomer. Z isomer: high-resolution mass spectrum (FAB) m/z 979.5902[(M+Na)+, calcd for C53 H84N2O13Na 979.5871].

[0275] 1.4. Rapamycin (E and Z)-24-(O-isobutyloxime) (9, 10)

[0276] Prepared in an analogous manner to Rapamycin (E andZ)-24-(O-methyloxime). The isomer mixture was separated by HPLC (15%H₂O/MeCN through a Kromasil C-18 250×20 mm column, 12 mL/min) to provide28 mg (65%) of the faster eluting Z isomer and 3.0 mg (7%) of the Eisomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1007.6146[(M+Na)+, calcd for C55 H88N2O13Na 1007.6184]. E isomer: high-resolutionmass spectrum (FAB) m/z 1007.6157 [(M+Na)+, calcd for C55H88N2O13Na1007.6184].

[0277] 1.5. Rapamycin (E and Z)-24-(O-benzyloxime) (11, 12)

[0278] Prepared in an analogous manner to Rapamycin (E andZ)-24-(O-methyloxime). The isomer mixture was separated by HPLC (15%H₂O/MeCN through a Kromasil C-18 250×20 mm column, 12 mL/min) to provide19.6 mg (44%) of the faster eluting Z isomer and 6.1 mg (14%) of the Eisomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1041.6033[(M+Na)+, calcd for C58 H86N2O13Na 1041.6028]. E isomer: high-resolutionmass spectrum (FAB) m/z 1041.5988 [(M+Na)+, calcd for C58H86N2O13Na1041.6028].

[0279] 1.6. Rapamycin (E and Z)-24-(O-carboxymethyloxime) (13, 14))

[0280] Prepared in an analogous manner to Rapamycin (E andZ)-24-(O-methyloxime). The isomer mixture was separated by HPLC (45%H₂O/MeCN through a Kromasil C-18 250×20 mm column, 12 mL/min) to provide4.6 mg (11%) of the faster eluting Z isomer and 1.0 mg (2%) of the Eisomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1009.5664[(M+Na)+, calcd for C53 H82N2O15Na 1009.5613]. E isomer: high-resolutionmass spectrum (FAB) m/z 1009.5604 [(M+Na)+, calcd for C53H82N2O15Na1009.5613].

[0281] 1.7. Rapamycin (E and Z)-24-(O-carboxamidomethyloxime) (15, 16)

[0282] Prepared in an analogous manner to Rapamycin (E andZ)-24-(O-methyloxime). The isomer mixture was separated by HPLC (35%H₂O/MeCN through a Kromasil C-18 250×20 mm column, 12 mL/min) to provide6.2 mg (10%) of the faster eluting Z isomer and 1.4 mg (2%) of the Eisomer. Z isomer: high-resolution mass spectrum (FAB) m/z 1008.5790[(M+Na)+, calcd for C53 H83N3O14Na 1008.5768]. E isomer: high-resolutionmass spectrum (FAB) m/z 1008.5753 [(M+Na)+, calcd for C53H83N3O14Na1008.5768].

Example 2 Assay of binding of rapamycin C24 derivatives to FKBP

[0283] Affinities of rapamycin C24 analogs for FKBP were determinedusing a competitive assay based on fluorescence polarization (FP). Afluorescein-labelled FK506 probe (AP1491) was synthesized, and theincrease in the polarization of its fluorescence used as a directreadout of % bound probe in an equilibrium binding experiment containingsub-saturating FKBP and variable amounts of rapamycin analog ascompetitor.

[0284] Synthesis of Fluoresceinated FK506 Probe (AP1491)

[0285] 2.1. 24, 32-Bis(tert-Butyldimethylsilyl)ether of FK506

[0286] tert-Butyldimethylsilyl trifluoromethanesulfonate (108 μL, 470μmol) was added dropwise to a stirred solution of FK506 (103 mg, 128μmol) and 2,6-lutidine (89.5 μL, 768 μmol) in dichloromethane (3 mL) at0° C. The resulting solution was stirred at 0° C. for 2 h, and thentreated with MeOH (0.5 mL) and ether (15 mL). The mixture was washedwith 10% aqueous NaHCO3 (3 mL) and brine (3 mL). The organic layer wasdecanted, dried over anhydrous Na2SO4, filtered, and concentrated to ayellow oil. Column chromatography (silica-gel, hexanes-EtOAc 3:1) gavethe title compound as a colorless oil (104 mg).

[0287] 2.2. Intermediate 1

[0288] To a solution of 24,32-bis(tert-butyldimethylsilyl)ether of FK506(100 mg, 97 μmol) in THF (2.5 mL) was added morpholine N-oxide (68 mg,580 μmol), followed by water (60 μL), and a 4% aqueous solution ofosmium tetroxide (123 μL, 20 μmol). The resulting mixture was stirred atroom temperature for 4.5 h. It was then treated with 50% aqueous MeOH(1.5 mL) and sodium periodate (207 mg, 97 μmol), and the suspensionstirred for an additional 1 h. The mixture was diluted with ether (10mL) and washed with saturated aqueous NaHCO3 (2×4 mL). The organic layerwas decanted, dried over anhydrous sodium sulfate containing a smallamount of sodium sulfite, filtered, and concentrated. The residue wasdissolved in anhydrous THF (2.8 mL), cooled to −78° C. under nitrogen,and treated with a 0.5 M solution of lithium tris[(3-ethyl-3-pentyl)oxy]aluminum hydride in THF (282 μL). The resultingsolution was stirred at −78° C. for 1.75 h, and then quenched byaddition of ether (6 mL) and saturated ammonium chloride solution (250μL). The mixture was allowed to warm up to room temperature and treatedwith anhydrous sodium sulfate. Filtration and concentration underreduced pressure afforded a pale yellow oil (97 mg), which was purifiedby column chromatography (silica-gel, hexanes-EtOAc 3:1) to afford 1 asa colorless oil.

[0289] 2.3 Intermediate 2

[0290] A solution of the above alcohol (300 mg, 290 μmol) inacetonitrile (10 mL) was treated with 2,6-lutidine (338 μL, 2.9 mmol)and N,N′-disuccinimidylcarbonate (371 mg, 1.45 mmol). The resultingsuspension was stirred at room temperature for 14.5 h, and thenconcentrated under reduced pressure. The residue was chromatographed(silica-gel, hexanes-EtOAc 2:1 to 100% EtOAc gradient) to afford themixed carbonate 2 as a pale yellow oil (127 mg).

[0291] 2.4 Intermediate 3

[0292] A solution of the above carbonate (30 mg, 26 μmol) andtriethylamine (36 μL, 260 μmol) in acetonitrile (1 mL) was treated with4′-(aminomethyl)fluorescein (13.5 mg, 34 μmol). The resulting brightorange suspension was stirred at room temperature for 1 h, and thenconcentrated under reduced pressure. The residue was chromatographed(silica-gel, hexanes-EtOAc 1:1 to 100% EtOAc to EtOAc-MeOH 1:1 gradient)to give 3 (20.5 mg) as a bright yellow solid.

[0293] 2.5 Compound 4

[0294] A solution of bis-silyl ether 3 (35 mg, 25 μmol) in acetonitrile(2 mL) was treated with 48% (w/w) HF in water (250 μL). The resultingmixture was stirred at room temperature for 5.5 h. It was then dilutedwith dichloromethane (10 mL) and washed with water (2×2 mL). The organiclayer was decanted, dried over anhydrous sodium sulfate, andconcentrated under reduced pressure. The residue was chromatographed(silica-gel, 100% EtOAc) to afford 4 (13 mg) as a bright yellow solid.

[0295] 2.6 Determination of Binding Affinities (IC50s) of Rapalogs UsingFP

[0296] Serial 10-fold dilutions of each analog were prepared in 100%ethanol in glass vials and stored on ice. All other manipulations wereperformed at room temperature. A stock of recombinant pure FKBP(purified by standard methods, see eg. Wiederrecht, G. et al. 1992. J.Biol. Chem. 267, 21753-21760) was diluted to approximately 3 nM in 50 mMpotassium phosphate pH 7.8/150 mM NaCl/100 μg/ml bovine gamma globulin(“FP buffer”: prepared using only low-fluorescence reagents fromPanvera) and 98 μl aliquots transferred to wells of a Dynatechmicro-fluor black 96-well fluorescence plate. 2.0 μl samples of therapamycin analogs were then transferred in duplicate to the wells withmixing. Finally, a probe solution was prepared containing 10 nM AP1491in 0.1% ethanol/FP buffer, and 100 μl added to each well with mixing.Duplicate control wells contained ethanol instead of rapamycin analog(for 100% probe binding) or ethanol instead of rapamycin analog and FPbuffer instead of FKBP (0% binding).

[0297] The plates were stored covered in the dark for approximately 30min to permit equilibration and then the fluorescence polarization ofthe sample in each well read on a Jolley FPM-2 FP plate reader JolleyConsulting and Research, Inc., Grayslake, Ill.) in accordance with themanufacturer's recommendations. The mean polarization (mP units) TABLE 4

fold loss in FKBPwt FP affinity binding assay (vs cmpd C24 isomer IC50(nM) rapamycin) rapamycin

2.3 (1) C14 desoxo

63.3 27.5 17

Z (major) 618 269 18

E (minor) 59.1 25.7  5

Z (major) 1416 616  6

E (minor) 438 190  7

Z (major) 2960 1287  8

E (minor) 1664 723  9

Z (major) >30000 >13043 10

E (minor) 2048 890 19

Z (major) >30000 >13043 20

E (minor) 2406 1046 11

Z (major) 8342 3627 12

E (minor) 1416 616 13

Z (major) 7960 3461 14

E (minor) 2351 1022 15

Z (major) 1151 500 16

E (minor) 204 88.7

[0298] for each competitor concentration was usually converted to %total binding by reference to the control values and plotted (y) vs. logmolar final concentration of competitor (x). Non-linear least squareanalysis was used to fit the curve and extract the IC50 using thefollowing equation:

y=M1+(M4−M1)/(1+exp(M2*(M3−x)))

[0299] where M3 is the IC50. For incomplete curves the IC50 wasdetermined by interpolation. Rapamycin and C14-desoxo-rapamycin wereincluded as controls in each case (C14-desoxo-rapamycin was prepared asdescribed by Luengo, J. I. et al. 1994 Tet Lett. 35, 6469-6472).

[0300] 2.7 Results of Binding Analysis of Rapamycin C24 Oximes

[0301] Affinities are reported as IC50s and as fold loss in affinity(=IC50/IC50 of rapamycin). (Comparative binding data of C24 rapalogs vsrapamycin and desoxo-rapamycin towards human FKBP12 are plotted inPCT/US86/09848.)

Example 3 Synthesis of C7 Rapalogs; Assay of Binding of C7 Rapalog-FKBPComplexes to FRAP

[0302] A series of C7 rapalogs containing various C7 substituentsselected from branched and unbranched alkoxy, arylalkyloxy,—NHCO-Oalkyl, —NHSO₂alkyl and substituted aryl and heteroaryl moietieswas synthesized using chemistry generally as described in the literatureexcept as noetd (see e.g., Luengo et al. 1995. Chemistry and Biology 2,471481, and the references cited in Table II for additional background).See also the table which follows.

[0303] 3.1 Compounds 27, 28—(R^(C7)=Et) are synthesized as described inLuengo et al, Chemistry & Biology July 1995, 2:471-481.

[0304] 3.2 Compound 29—(R^(C7)=iPr) A solution of rapamycin (60 mg,0.066 mmol) in 2-propanol (3 mL) at room temperature was treated withpara-toluenesulfonic acid (75 mg, 0.394 mmol) and allowed to stir for 4h. After this time the reaction was poured onto a biphasic solution ofsaturated aqueous NaHCO₃ (20 mL) and EtOAc (30 mL). The organic layerwas washed with additional solution of saturated aqueous NaHCO₃ (2×20mL) followed by a saturated aqueous solution of NaCl (2×10 mL) thendried over Na₂SO₄, filtered, evaporated. The resulting material waspurified by HPLC on a Kromasil C-18 column (20×250 mm) at 55 C using 65%acetonitrile/water as eluant to afford AP1700 (25 mg). MS(FAB):(M+Na)+calcd: 964.5762, found: 964.5753.

[0305] 3.3 Compound 30—(R^(C7)=benzyl) is synthesized as described inChemistry & Biology July 1995, 2:471-481.

[0306] 3.4 Compounds 31, 32—(R^(C7)=—NH—CO—OMe) may be synthesized asdescribed in Chemistry & Biology July 1995, 2:471-481.

[0307] 3.5 Compound 33—(R^(C7)=—NH—SO₂—Me) A solution of rapamycin (75mg, 0.082 mmol) and methanesufonamide (312 mg, 3.282 mmol) indichloromethane (3 mL) at −40° C. was treated dropwise withtrifluoroacetic acid (126 μL, 1.636 mmol) and allowed to stir for 3 h.After this time the reaction was poured onto a biphasic solution ofsaturated aqueous NaHCO₃ (20 mL) and EtOAc (10 mL). The organic layerwas washed with a saturated aqueous solution of NaCl (2×10 mL) thendried over Na₂SO₄, filtered, evaporated, and flash chromatographed on asilica gel (dichloromethane:hexane:EtOAc: MeOH, 200:50:42.5:7.5). Theresulting semipurified material was purified by HPLC on a Kromasil C-18column (20×250 mm) at 55 C using 65% acetonitrile/water as eluant toafford AP1705 (24 mg). MS(FAB): (M+Na)+calcd: 999.5246, found: 999.5246.

[0308] 3.6 Compounds 34, 35—(R^(C7)=furanyl) These compounds may besynthesized as described in Chemistry & Biology July 1995, 2:471-481.

[0309] 3. Compounds 36, 37—(R^(C7)=methylthiophene) These compounds maybe synthesized as described in J. Org. Chem 1994, 59, 6512-6513.

[0310] 3.8 Compounds 39, 38—(R^(C7)=ethylthiophene) A solution ofrapamycin (50 mg, 0.055 mmol) and 2-ethylthiophene (248 μL, 2.188 mmol)in dichloromethane (1.5 mL) at −40° C. was treated dropwise withtrifluoroacetic acid (84 uL, 1.094 mmol) and allowed to stir for 3 h.After this time the reaction was poured onto a biphasic solution ofsaturated aqueous NaHCO₃ (15 mL) and EtOAc (10 mL). The organic layerwas washed with a saturated aqueous solution of NaCl (2×10 mL) thendried over Na₂SO₄, filtered, evaporated, and flash chromatographed on asilica gel (MeOH:dichloromethane, 2:98 then 5:95). The resultingsemipurified material was purified by HPLC on a Kromasil C-18 column(20×250 mm) at 55 C

# R^(C7a) R^(C7b) rapamycin —OMe H C14- —OMe H desoxo rapamycin 27 —OEtH 28 H —OEt 29 —O-iPr H 30 —O-benzyl H 32 —NH—(C═O)—OMe H 31 H—NH—(C═O)—OMe 33 —NH—SO₂Me H 34

H 35 H

36

H 37 H

38

H 39 H

41

H 40 H

42 -o,p- H dimethoxyphenyl 43 H -o,p- dimethoxyphenyl 44

H 45 H

46 -o,p- H diethoxyphenyl 47

H 48

H 49 -2,4,6- H trimethoxyphenyl 50 H -2,4,6- trimethoxyphenyl 51—NH—(C═O)—OEt H 52 H —NH—(C═O)—OEt

[0311] using 80% acetonitrile/water as eluant to afford AP1858 (6 mg)and AP1859 (28 mg). MS(ES+): (M+NH₄)⁺ 1016; MS(ES—): (M−H)-992.

[0312] 3.9 Compounds 40, 41—(R^(C7)=tertbutyl thiophene) A solution ofrapamycin (50 mg, 0.055 mmol) and 2-tert-butylylthiophene (276 mg, 2.188mmol) in dichloromethane (1.5 mL) at 40° C. was treated dropwise withtrifluoroacetic acid (84 μL, 1.094 mmol) and allowed to stir for 3 h.After this time the reaction was poured onto a biphasic solution ofsaturated aqueous NaHCO₃ (15 mL) and EtOAc (10 mL). The organic layerwas washed with a saturated aqueous solution of NaCl (2×10 mL) thendried over Na₂SO₄, filtered, evaporated, and flash chromatographed on asilica gel (MeOH:dichloro-methane, 2:98 then 5:95). The resultingsemipurified material was purified by HPLC on a Kromasil C-18 column(20×250 mm) at 55 C using 80% acetonitrile/water as eluant to affordAP1856 (4 mg) and AP1857 (14 mg). MS(ES+): (M+Na)⁺ 1045; MS(ES−): (M−H)⁻1021.

[0313] 3.10 Compounds 43, 42—(R^(C7)=o,p-dimethoxyphenyl) Thesecompounds may be found in Chemistry & Biology July 1995, 2:471-481.

[0314] 3.11 Compounds 44, 45-(R^(C7)=indolyl) A solution of rapamycin(50 mg, 0.055 mmol) and indole (64 mg, 0.547 mmol) in dichloromethane(2.0 mL) at −40° C. was treated dropwise with trifluoroacetic acid (84μL, 1.094 mmol) and allowed to stir for 3 h. After this time thereaction was poured onto a biphasic solution of saturated aqueous NaHCO₃(15 mL) and EtOAc (10 mL). The organic layer was washed with a saturatedaqueous solution of NaCl (2×10 mL) then dried over Na₂SO₄, filtered,evaporated, and flash chromatographed on a silica gel(dichloromethane:hexane:EtOAc: MeOH, 200:50:42.5:7.5). The resultingsemipurified material was purified by HPLC on a Kromasil C-18 column(20×250 mm) using 65% acetonitrile/water as eluant for AP1701 (12 mg)and AP1702 (7.6 mg). MS(FAB): (M+Na)^(+ calcd:) 1021.5765, found:1021.5788 (AP1701) and 1021.5797 (AP1702).

[0315] 3.12 Compound 46—(R^(C7)=o,p-diethoxyphenyl) A solution ofrapamycin (108 mg, 0.118 mmol) and 1,3-diethoxybenzene (783 mg, 4.72mmol) in dichloromethane (2.0 mL) at −40° C. was treated dropwise withtrifluoroacetic acid (154 μL, 2.01 mmol) and allowed to stir for 3 h.After this time the reaction was poured onto a biphasic solution ofsaturated aqueous NaHCO₃ (15 mL) and EtOAc (15 mL). The organic layerwas washed with a saturated aqueous solution of NaCl (2×10 mL) thendried over Na₂SO₄, filtered, evaporated, and flash chromatographed on asilica gel (dichloromethane:hexane:EtOAc:MeOH, 200:50:42.5:7.5). Theresulting material was purified by HPLC on a Rainin silica column(20×250 mm) using (dichloromethane:hexane:EtOAc:MeOH, 210:65:65:10) aseluant for AP20808 (20 mg). MS(ES+): (M+Na)+1065.95.

[0316] 3.13 Compound 47—(R^(C7)=methylthiophene) A solution of rapamycin(105 mg, 0.115 mmol) and 3-methylthiophene(445 μL, 4.60 mmol) indichloromethane (2.0 mL) at −40° C. was treated dropwise withtrifluoroacetic acid (150 μL, 1.96 mmol) and allowed to stir for 3 h.After this time the reaction was poured onto a biphasic solution ofsaturated aqueous NaHCO₃ (15 mL) and EtOAc (15 mL). The organic layerwas washed with a saturated aqueous solution of NaCl (2×10 mL) thendried over Na₂SO₄, filtered, evaporated, and flash chromatographed on asilica gel (dichloromethane:hexane:EtOAc:MeOH, 200:50:42.5:7.5). Theresulting material was purified by HPLC on a Rainin silica column(20×250 mm) using (dichloromethane:hexane:EtOAc:MeOH, 210:65:65:10) aseluant for AP20809 (60 mg). MS(ES+): (M+Na)⁺ 1002.96.

[0317] 3.14 Compound 48-(R^(C7)=N-methylpyrrole) A solution of rapamycin(51 mg, 0.056 mmol) and N-methylpyrrole (198 μL 2.23 mmol) indichloromethane (2.0 mL) at 0° C. was treated with zinc chloride (76 mg,0.557 mmol) and allowed to warm to rt overnight. After this time thereaction was poured onto a biphasic solution of saturated aqueous NaHCO₃(15 mL) and EtOAc (15 mL). The organic layer was washed with a saturatedaqueous solution of NaCl (2×10 mL) then dried over Na₂SO₄, filtered,evaporated, and flash chromatographed on a silica gel(dichloromethane:hexane:EtOAc:MeOH, 100:150:150:10). The resultingmaterial was purified by HPLC on a Rainin Si column (20×250 mm) using(dichloromethane:hexane:EtOAc:MeOH, 210:65:65:10) as eluant for AP20810(10 mg). MS(ES+): (M+NH4)⁺ 981.05; MS(ES−): (M−H)⁻ 961.69.

[0318] The C7 rapalogs were characterized by exact mass spec and NMR.

[0319] 3.15 Assay of FKBP Binding Affinity of C7 Rapalogs

[0320] The affinity of a variety of the C7 rapalogs for FKBP was assayedas described for C24 rapalogs above, using competitive FP. Rapamycin andC14-desoxo-rapamycin (prepared as described by Luengo et al. 1994.Tetrahedron Lett. 35, 6469-6472) were included as controls.

[0321] Affinities are reported below as IC50s and fold loss in affinity(═IC50/IC50 of rapamycin). See “Illustrative C7 Rapalogs” Table below.These data indicate that these large C7 substituents do not necessarilycause large reductions in the affinity of the rapalogs for human FKBP.FKBPwt FP fold loss in binding assay affinity (cf Compound IC50 (nM)rapamycin) rapamycin 2.3 (1) C14 desoxorap 34 15 27 2.6 1.1 28 3.7 1.629 2.2 1.0 30 12 5.2 32 4.3 1.9 31 2.6 1.1 33 2.5 1.1 34 28 12 35 29 1336 3.7 1.6 37 4.3 1.9 38 2.5 1.1 39 2.4 1.0 41 2.9 1.3 40 3.4 1.5 42 2.21.0 43 20 8.7 44 7.8 3.3 45 5.9 2.6

Example 4 Preparation of Rapalogs Modified at R^(C24) and R^(C30):24(S),30(S)-tetrahydro-rapamycin (53)

[0322]

[0323] Rapamycin (46 mg, 0.050 mmol) was dissolved in 2.0 mL ofmethanol, cooled to −78° C., and cerium (III) chloride heptahydrate (46mg, 0.123 mmol) was added. The solution was stirred for 0.25 h., thensodium borohydride (7.6 mg, 0.20 mmol) was added. After 0.5 h, thereaction mixture was partitioned between ethyl acetate (15 mL) and 5%aqueous hydrochloric acid (2 mL). The organic phase was washed withwater (2 mL) and brine (1 mL), dried over anhydrous magnesium sulfate,filtered, and concentrated. Flash chromatography (silica gel,15:75:50:200 methanol:ethyl acetate:hexane:dichloromehane) yielded 35 mg(76%) of the desired product as a white foam. Mass spectral data:(ES+/NaCl/NH3) m/z 942.21 (M+Na)+, 935.83 (M+NH4)+; (ES−/NaCl) m/z963.04 (M+Cl)−, 917.34 (M−H)− lit. ref. Luengo, J. I.; Rozamus, L. W.;Holt, D. A. Tetrahedron Lett. 1994, 35, 6469-6472.

Example 5 Preparation of Rapalogs Modified at C24, C30 and C7

[0324] 24(S), 30(S)-tetrahydrorapamycin (53), prepared as in Example 4,may be modified at C7 using approaches illustrated in the prior C7rapalog examples. For example:

[0325] 5.1 7(S)-(2′,4′-dimethoxy)benzyl-7-demethoxy-24(S),30(S)-tetrahydro-rapamycin

[0326] 24(S), 30(S)-tetrahydro-rapamycin (20 mg, 0.022 mmol) wasdissolved in dichloro-methane (1.0 mL). 1,3-dimethoxybenzene (0.20 mL,1.5 mmol) was added, and the solution was cooled to −60° C.Trifluoroacetic acid (0.030 mL, 0.39 mmol) was added, and the reactionmixture was stirred for 1 h at −60° C., then partitioned between ethylacetate (10 mL) and saturated aqueous sodium bicarbonate (1 mL). Theorganic phase was washed with water (2 mL) and brine (1 mL), dried overanhydrous magnesium sulfate, filtered, and concentrated. Flashchromatography (silica gel, 15:75:50:200 methanol: ethylacetate:hexane:dichloromehane) yielded 8 mg (35%) of the desired productas a white solid. Mass spectral data: (ES+/NaCl/NH3) m/z 1046.96(M+Na)+, 1042.15 (M+NH4)+; (ES−/NaCl) m/z 1069.09 (M+Cl)− lit. ref.Luengo, J. I.; Konialian-Beck, A.; Rozamus, L. W.; Holt, D. A. J. Org.Chem. 1994, 59, 6512-6513.

[0327] By analogous means, one may produce 24(S), 30(S)-tetrahydrorapamycins bearing other C7 substituents as described elsewhere herein,e.g., containing alternatively substituted aryl groups, heteroaryl,—O-aliphatic groups, thioethers, or any of the other types of moietiesdesignated previously for R^(C7a) or R^(C7b). These compounds may beobtained by reduction at C24 and C30 of the appropriate C7 rapalog, orby transformation at C7 of the appropriate C24, C30-tetrahydro rapalog.Illustrative examples follow.

[0328] Rapalogs modified at C24, C30 and C7 may also be differ fromrapamycin at the various positions discussed herein, e.g. with respectto one or more of R^(C13), R^(C43), R^(C28), R^(C29) R⁴, “a” etc. By wayof example, starting with 13-F-rapamycin in place of rapamycin yieldsthe 13-fluoro analogs of compounds 53-79.

[0329] 5.2 Compounds 54, 55—(R^(C7)=Et) are synthesized as described inExample 4.1, but substituting Compounds 27 and 28, respectively, forrapamycin.

[0330] 5.3 Compound 56—(R^(C7)=iPr) is synthesized as described inExample 4.1, but substituting Compound 29 for rapamycin.

[0331] 5.4 Compound 57-(R^(C7)=benzyl) is synthesized as described inExample 4.1, but substituting Compound 30 for rapamycin.

[0332] 5.5 Compounds 58, 59—(R^(C7)=—NH—CO—OMe) are synthesized asdescribed in Example 4.1, but substituting Compounds 32 and 31,respectively, for rapamycin.

[0333] 5.6 Compound 60—(R^(C7)=—NH—SO2-Me) is synthesized as describedin Example 4.1, but substituting Compound 33 for rapamycin

[0334] 5.7 Compounds 61 and 62—(R^(C7)=furanyl) are synthesized asdescribed in Example 4.1, but substituting Compounds 34 and 35respectively, for rapamycin.

[0335] 5.8 Compounds 63, 64—(R^(C7)=methylthiophene) are synthesized asdescribed in Example 4.1, but substituting Compounds 36 and 37,respectively, for rapamycin.

[0336] 5.9 Compounds 65, 66—(R^(C7)=ethylthiophene) are synthesized asdescribed in Example 4.1, but substituting Compounds 38 and 39,respectively, for rapamycin.

[0337] 5.10 Compounds 67, 68—(R^(C7)=tertbutyl thiophene) aresynthesized as described in Example 4.1, but substituting Compounds 41and 40, respectively, for rapamycin.

[0338] 5.11 Compounds 69, 70—(R^(C7)=o,p-dimethoxyphenyl) aresynthesized as described in Example 4.1, but substituting Compounds 43and 42 respectively, for rapamycin.

[0339] 5.12 Compounds 71, 72-(R^(C7)=indolyl) are synthesized asdescribed in Example 4.1, but substituting Compounds 45 and 46,respectively, for rapamycin.

[0340] 5.13 Compound 73—(R^(C7)=o,p-diethoxyphenyl) is synthesized asdescribed in Example 4.1, but substituting Compound 46 for rapamycin.

[0341] 5.14 Compound 74—(R^(C7)=methylthiophene) is synthesized asdescribed in Example 4.1, but substituting Compound 47 for rapamycin.

# R^(C7a) R^(C7b) 53 —OMe H 54 —OEt H 55 H —OEt 56 —O-iPr H 57 —O-benzylH 58 —NH—(C═O)—OMe H 59 H —NH—(C═O)—OMe 60 —NH—SO₂Me H 61

H 62 H

63

H 64 H

65

H 66 H

67

H 68 H

69 -o,p-(MeO)₂phenyl H 70 H -o,p-(MeO)₂phenyl 71

H 72 H

73 -o,p-diethoxyphenyl H 74

H 75

H 76 -2,4,6-(MeO)₃phenyl H 77 H -2,4,6-(MeO)₃phenyl 78 —NH—(C═O)—OEt H79 H —NH—(C═O)—OEt

[0342] 5.15 Compound 75-(R^(C7)=N-methylpyrrole) is synthesized asdescribed in Example 4.1, but substituting Compound 48 for rapamycin.

[0343] 5.16 Compound 75, 76-(R^(C7=2,4,6)-trimethoxyphenyl) issynthesized as described in Example 5.1, but substituting1,3,5-trimethoxybenzene for 1,3-dimethoxybenzene.

Example 6 Preparation of Fluoro-rapalogs

[0344] 6.1 C13-Fluoro-rapalogs

[0345] A new class of rapalogs, C13-Fluoro-rapalogs, may be prepared bythe following route:

[0346] In this example, the hydroxyl moieties at positions 28 and 43 areprotected prior to treatment with DAST. We have used bis-triethylsilyl(as shown above) and bis-triisopropylsilyl protecting groups. Variousalternative protecting groups may be used, based on user preference orconvenience and in consideration of the reaction conditions ofsubsequent transformations prior to or concurrent with removal ofprotecting groups. The protected compound is then treated with the DASTreagent to introduce the 13-fluoro substituent. The DAST reaction may beconducted, e.g., at −42° C. as shown, or at 0° C.

[0347] 13-Fluoro rapamycin may then be modified at position 7 as desiredto produce the family of 13-fluoro C7-rapalogs bearing any of thevariety of moieties designated previously for R^(C7a) or R^(C7b.) Forinstance, the 7-(o,p-dimethoxy)-13-fluoro-rapalogs (96 and 97) may beprepared (and separately recovered if desired) by transformation of 78at C7 followed by removal of protecting groups, or, as shown below, byremoval of protecting groups from 78 followed by transformation at C7.

[0348] One may subject 13-F-rapamycin, instead of rapamycin, to variousother chemical transformations such as are disclosed or referred toherein, including, for instance, fluorination at C28, reduction at C24and C30, fluorination at C24 and C30, modification at C-43, etc., inaddition to or as an alternative to modification at C7, in order toobtain the corresponding 13-F analog.

[0349] 6.2 Compounds 81, 82—(R^(C7)=Et) are synthesized as described inExample 3.1, but substiuting 13-F-rapamycin (79) for rapamycin.

[0350] 6.3 Compound 83—(R^(C7)=iPr) is synthesized as described inExample 3.2, but substituting 13-F-rapamycin (79) for rapamycin.

[0351] 6.4 Compound 84-(R^(C7)=benzyl) is synthesized as described inExample 3.3, but substituting 13-F-rapamycin (79) for rapamycin.

[0352] 6.5 Compounds 8586—(R^(C7)=—NH—CO—OMe) are synthesized asdescribed in Example 3.4, but substituting 13-F-rapamycin (79) forrapamycin.

# R^(C7a) R^(C7b) 81 —OEt H 82 H —OEt 83 —O-iPr H 84 —O-benzyl H 85—NH—(C═O)—OMe H 86 H —NH—(C═O)—OMe 87 —NH—SO₂Me H 88

H 89 H

90

H 91 H

92

H 93 H

94

H 95 H

96 -o,p-(MeO)₂phenyl H 97 H -o,p-(MeO)₂phenyl 98

H 99 H

100 -o,p-diethoxyphenyl H 101

H 102

H 103 -2,4,6-(MeO)₃phenyl H 104 H -2,4,6-(MeO)₃phenyl 105 —NH—(C═O)—OEtH 106 H —NH—(C═O)—OEt

[0353] 6.6 Compound 87—(R^(C7)=—NH—SO2-Me) is synthesized as describedin Example 3.5, but substituting 13-F-rapamycin (79) for rapamycin.

[0354] 6.7 Compounds 88 and 89—(R^(C7)=furanyl) are synthesized asdescribed in Example 3.6, but substituting 13-F-rapamycin (79) forrapamycin.

[0355] 6.8 Compounds 9091—(R^(C7)=methylthiophene) are synthesized asdescribed in Example 3.7, but substituting 13-F-rapamycin (79) forrapamycin.

[0356] 6.9 Compounds 9293—(R^(C7)=ethylthiophene) are synthesized asdescribed in Example 3.8, but substituting 13-F-rapamycin (79) forrapamycin.

[0357] 6.10 Compounds 68, 69—(R^(C7)=tertbutyl thiophene) aresynthesized as described in Example 3.9, but substituting 13-F-rapamycin(79) for rapamycin.

[0358] 6.11 Compounds 94, 95—(R^(C7)=o,p-dimethoxyphenyl) aresynthesized as described in Example 3.10, but substituting13-F-rapamycin (79) for rapamycin.

[0359] 6.12 Compounds 9697-(R^(C7)=indolyl) are synthesized as describedin Example 3.11, but substituting 13-F-rapamycin (79) for rapamycin.

[0360] 6.13 Compound 98—(R^(C7)=o,p-diethoxyphenyl) is synthesized asdescribed in Example 3.12, but substituting 13-F-rapamycin (79) forrapamycin.

[0361] 6.14 Compound 99—(R^(C7)=methylthiophene) is synthesized asdescribed in Example 3.13, but substituting 13-F-rapamycin (79) forrapamycin.

[0362] 6.15 Compound 100-(R^(C7)=N-methylpyrrole) is synthesized asdescribed in Example 3.14, but substituting 13-F-rapamycin (79) forrapamycin.

[0363] 6.20 Preparation of 28-F-rapamycin (107)

[0364] To a solution of rapamycin (71 mg, 0.078 mmol) in methylenechloride (1 mL) at −78° C. was added DAST (21 mL, 0.156 mmol) andreaction was allowed to stir for 2 h before MeOH was added to quench thereaction. The reaction mixture was taken to room temperature and stirredfor 30 min. It was poured onto a biphasic solution of saturated aqueousNaHCO3 (20 mL) and EtOAc (30 mL). The organic layer was washed withadditional solution of saturated aqueous NaHCO3 (2×20 mL) followed by asaturated aqueous solution of NaCl (2×10 mL) then dried over Na2SO4,filtered, evaporated. The resulting material was flash chromatographedon a silica gel (hexane:EtOAc, 1:1 to 1:2). MS, Fluorine NMR indicatedC28 fluorinated rapamycin. Stereoisomers can be separated by reversephase chromatography (C18 column, MeOH:H2O, 80:20), and may be used inplace of rapamycin for the synthesis of various F-28 rapalogs.

[0365] 6.21 Compound 108-(13-F, 28-F-rapamycin) is synthesized asdescribed above for 28-F-rapamycin, but with twice the volume of DAST(41 mL) at a higher temperature (−40° C.).

Example 7 Constructs Encoding Chimeric Transcription Factors

[0366] A. Unless otherwise stated, all DNA manipulations described inthis and other examples were performed using standard procedures (Seee.g., F. M. Ausubel et al., Eds., Current Protocols in Molecular BiologyUohn Wiley & Sons, New York, 1994).

[0367] B. Plasmids

[0368] Constructs encoding fusions of human FKBP12 with the yeast GAL4DNA binding domain, the HSV VP16 activation domain, human T cell CD3zeta chain intracellular domain or the intracellular domain of human FASare disclosed in PCT/US94/01617.

[0369] Additional DNA vectors for directing the expression of fusionproteins relevant to this invention were derived from the mammalianexpression vector pCGNN (Attar, R. M. and Gilman, M. Z. 1992. MCB 12:2432-2443). Inserts cloned as XbaI-BamHI fragments into pCGNN aretranscribed under the control of the human CMV promoter and enhancersequences (nucleotides −522 to +72 relative to the cap site), and areexpressed with an optional epitope tag (a 16 amino acid portion of theH. influenzae hemaglutinin gene that is recognized by the monoclonalantibody 12CA5) and, in the case of transcription factor domains, withan N-terminal nuclear localization sequence (NLS; from SV40 T antigen).

[0370] Except where stated, all fragments cloned into pCGNN wereinserted as XbaI-BamHI fragments that included a SpeI site just upstreamof the BamHI site. As XbaI and SpeI produce compatible ends, thisallowed further XbaI-BamHI fragments to be inserted downstream of theinitial insert and facilitated stepwise assembly of proteins comprisingmultiple components. A stop codon was interposed between the SpeI andBamHI sites. For initial constructs, the vector pCGNN-GAL4 wasadditionally used, in which codons 1-94 of the GAL4 DNA binding domaingene were cloned into the XbaI site of pCGNN such that a XbaI site isregenerated only at the 3′ end of the fragment. Thus XbaI-BamHIfragments could be cloned into this vector to generate GAL4 fusions, andsubsequently recovered.

[0371] (a) Constructs Encoding GAL4 DNA Binding Domain-FRAP Fusions

[0372] To obtain portions of the human FRAP gene, human thymus total RNA(Clontech #64028-1) was reverse transcribed using MMLV reversetranscriptase and random hexamer primer (Clontech 1st strand synthesiskit). This cDNA was used directly in a PCR reaction containing primers 1and 2 and Pfu polymerase (Stratagene). The primers were designed toamplify the coding sequence for amino acids 2025-2113 inclusive of humanFRAP: an 89 amino acid region essentially corresponding to the minimal‘FRB’ domain identified by Chen et al. (Proc. Natl. Acad. Sci. USA(1995) 92, 4947-4951) as necessary and sufficient for FKBP-rapamycinbinding (hereafter named FRB). The appropriately-sized band waspurified, digested with XbaI and SpeI, and ligated into XbaI-SpeIdigested pCGNN-GAL4. This construct was confirmed by restrictionanalysis (to verify the correct orientation) and DNA sequencing anddesignated pCGNN-GAL4-1FRB.

[0373] Constructs encoding FRB multimers were obtained by isolating theFRB XbaI-BamHI fragment, and then ligating it back into pCGNN-GAL4-1FRBdigested with SpeI and BamHI to generate pCGNN-GAL4-2FRB, which wasconfirmed by restriction analysis. This procedure was repeatedanalogously on the new construct to yield pCGNN-GAL4-3FRB andpCGNN-GAL4-4FRB.

[0374] Vectors were also constructed that encode larger fragments ofFRAP, encompassing the minimal FRB domain (amino acids 2025-2113) butextending beyond it. PCR primers were designed that amplify variousregions of FRAP flanked by 5′ XbaI and 3′ SpeI sites as indicated below.Designation amino acid 5′ primer 3′ primer FRAPa 2012-2127 6 7 FRAPb1995-2141 5 8 FRAPc 1945-2113 3 2 FRAPd 1995-2113 5 2 FRAPe 2012-2113 62 FRAPf 2025-2127 1 7 FRAPg 2025-2141 1 8 FRAPh 2025-2174 1 4 FRAPi1945-2174 3 4

[0375] Initially, fragment FRAPi was amplified by RT-PCR as describedabove, digested with XbaI and SpeI, and ligated into XbaI-SpeI digestedpCGNN-GAL4. This construct, pCGNN-GAL4-FRAPi, was analyzed by PCR toconfirm insert orientation and verified by DNA sequencing. It was thenused as a PCR substrate to amplify the other fragments using the primerslisted. The new fragments were cloned as GAL4 fusions as described aboveto yield the constructs pCGNN-GAL4-FRAPa, pCGNN-GAL4-FRAPb etc, whichwere confirmed by DNA sequencing.

[0376] Vectors encoding concatenates of two of the larger FRAPfragments, FRAPd and FRAPe, were generated by analogous methods to thoseused earlier. XbaI-BamHI fragments encoding FRAPd and FRAPe wereisolated from pCGNN-GAL4-FRAPd and pCGNN-GAL4-FRAPe and ligated backinto the same vectors digested with SpeI and BamHI to generatepCGNN-GAL4-2FRAPd and pCGNN-GAL4-2FRAPe. This procedure was repeatedanalogously on the new constructs to yield pCGNN-GAL4-3FRAPd,pCGNN-GAL4-3FRAPe, pCGNN-GAL4-4FRAPd and pCGNN-GAL4-4FRAPe. Allconstructs were verified by restriction analysis.

[0377] (b) Constructs Encoding FRB-VP16 Activation Domain Fusions

[0378] To generate N-terminal fusions of FRB domain(s) with theactivation domain of the Herpes Simplex Virus protein VP16, theXbaI-BamHI fragments encoding 1, 2, 3 and 4 copies of FRB were recoveredfrom the GAL4 fusion vectors and ligated into XbaI-BamHI digested pCGNNto yield pCGNN-1FRB, pCGNN-2FRB etc. These vectors were then digestedwith SpeI and BamHI. An XbaI-BamHI fragment encoding amino acids 414-490of VP16 was isolated from plasmid pCG-Gal4-VP16 (Das, G., Hinkley, C. S.and Herr, W. (1995) Nature 374, 657-660) and ligated into the SpeI-BamHIdigested vectors to generate pCGNN-1FRB-VP16, pCGNN-2FRB-VP16, etc. Theconstructs were verified by restriction analysis and/or DNA sequencing.

[0379] (c) Constructs Encoding ZFHD1 DNA Binding Domain-FRB Fusions

[0380] An expression vector for directing the expression of ZFHD1 codingsequence in mammalian cells was prepared as follows. Zif268 sequenceswere amplified from a cDNA clone by PCR using primers 5′Xba/Zif and3′Zif+G. Oct1 homeodomain sequences were amplified from a cDNA clone byPCR using primers 5′Not Oct HD and Spe/Bam 3′Oct. The Zif268 PCRfragment was cut with XbaI and NotI. The Oct1 PCR fragment was cut withNotI and BamHI. Both fragments were ligated in a 3-way ligation betweenthe XbaI and BamHI sites of pCGNN (Attar and Gilman, 1992) to makepCGNNZFHD1 in which the cDNA insert is under the transcriptional controlof human CMV promoter and enhancer sequences and is linked to thenuclear localization sequence from SV40 T antigen. The plasmid pCGNNalso contains a gene for ampicillin resistance which can serve as aselectable marker. (Derivatives, pCGNNZFHD1-FKBPx1 andpCGNNZFHD1-FKBPx3, were prepared containing one or three tandem repeatsof human FKBP12 ligated as an XbaI-BamHI fragment between the SpeI andBamHI sites of pCGNNZFHD1. A sample of pCGNNZFHD1-FKBPx3 has beendeposited with the American Type Culture Collection under ATCC AccessionNo. 97399. Sequences of primers is shown in WO 96/41865.

[0381] To generate C-terminal fusions of FRB domain(s) with the chimericDNA binding protein ZFHD1, the XbaI-BamHI fragments encoding 1, 2, 3 and4 copies of FRB were recovered from the GAL4 fusion vectors and ligatedinto Spe-BamHI digested pCGNN-ZFHD1 to yield pCGNN-ZFHD1-1FRB,pCGNN-ZFHD1-2FRB etc. Constructs were verified by restriction analysisand/or DNA sequencing.

[0382] To examine the effect of introducing additional ‘linker’polypeptide between ZFHD1 and a C-terminal FRB domain, FRAP fragmentsencoding extra sequence N-terminal to FRB were cloned as ZFHD1 fusions.XbaI-BamHI fragments encoding FRAPa, FRAPb, FRAPc, FRAPd and FRAPe wereexcised from the vectors pCGNN-GAL4-FRAPa, pCGNN-GAL4-FRAPb etc andligated into SpeI-BamHI digested pCGNN-ZFHD1 to yield the vectorspCGNN-ZFHD1-FRAPa, pCGNN-ZFHD1-FRAPb, etc. Vectors encoding fusions ofZFHD1 to 2, 3 and 4 C-terminal copies of FRAPe were also constructed byisolating XbaI-BamHI fragments encoding 2FRAPe, 3FRAPe and 4FRAPe frompCGNN-GAL4-2FRAPe, pCGNN-GAL4-3FRAPe and pCGNN-GAL4-4FRAPe and ligatingthem into SpeI-BamHI digested pCGNN-ZFHD1 to yield the vectorspCGNN-ZFHD1-2FRAPe, pCGNN-ZFHD1-3FRAPe and pCGNN-ZFHD1-4FRAPe. Allconstructs were verified by restriction analysis.

[0383] Vectors were also constructed that encode N-terminal fusions ofFRB domain(s) with ZFHD1. XbaI-BamHI fragments encoding 1, 2, 3 and 4copies of FRAPe were isolated from pCGNN-GAL4-1FRAPe, pCGNN-GAL4-2FRAPeetc and ligated into XbaI-BamHI digested pCGNN to yield the plasmidspCGNN-1FRAPe, pCGNN-2FRAPe etc. These vectors were then digested withSpeI and BamHI, and an XbaI-BamHI fragment encoding ZFHD1 (isolated frompCGNN-ZFHD1) ligated in to yield the constructs pCGNN-1FRAPe-ZFHD1,pCGNN-2FRAPe-ZFHD1 etc, which were verified by restriction analysis.

[0384] (d) Constructs Encoding FRB-p65 Activation Domain Fusions

[0385] To generate fusions of FRB domain(s) with the activation domainof the human NF-kB p65 subunit (hereafter designated p65), two fragmentswere amplified by PCR from the plasmid pCG-p65. Primers 9 (p65/5′ Xba)and 11 (p65 3′ Spe/Bam) amplify the coding sequence for amino acids450-550, and primers 10 (p65/36′ Xba) and 11 amplify the coding sequencefor amino acids 361-550, both flanked by 5′ XbaI and 3′ SpeI/BamHIsites. PCR products were digested with XbaI and BamHI and cloned intoXbaI-BamHI digested pCGNN to yield pCGNN-p65(450-550) andpCGNN-p65(361-550). The constructs were verified by restriction analysisand DNA sequencing.

[0386] DNA sequences encoding the 100 amino acid P65 transcriptionactivation sequence and the more extended p65 transcription activationdomain (351-550) are shown in WO 96/41865.

[0387] To generate N-terminal fusions of FRB domain(s) with portions ofthe p65 activation domain, plasmids pCGNN-1FRB, pCGNN-2FRB etc weredigested with SpeI and BamHI. An XbaI-BamHI fragment encoding p65(450-550) was isolated from pCGNN-p65(450-550) and ligated into theSpeI-BamHI digested vectors to yield the plasmidspCGNN-1FRB-p65(450-550), pCGNN-2FRB-p65(450-550) etc. The constructpCGNN-1FRB-p65(361-550) was made similarly using an XbaI-BamHI fragmentisolated from pCGNN-p65(361-550). These constructs were verified byrestriction analysis.

[0388] To examine the effect of introducing additional ‘linker’polypeptide between the p65 activation domain and an N-terminal FRBdomain, FRAP fragments encoding extra sequence C-terminal to FRB werecloned as p65 fusions. XbaI-BamHI fragments encoding FRAPa, FRAPb,FRAPf, FRAPg and FRAPh were excised from the vectors pCGNN-GAL4-FRAPa,pCGNN-GAL4-FRAPb etc and ligated into XbaI-BamHI digested pCGNN to yieldthe vectors pCGNN-FRAPa, pCGNN-FRAPb, etc. These plasmids were thendigested with SpeI and BamHI, and a XbaI-BamHI fragment encoding p65(amino acids 450-550) ligated in to yield the five vectorspCGNN-FRAPa-p65, pCGNN-FRAPb-p65, etc, which were verified byrestriction analysis.

[0389] Vectors encoding fusions of p65 to 1 and 3 N-terminal copies ofFRAPe were also prepared by digesting pCGNN-1FRAPe and pCGNN-3FRAPe withSpeI and BamHI. XbaI-BamHI fragments encoding p65(450-550) andp65(361-550) (isolated from pCGNN-p65(450-550) and pCGNN-p65(361-550))were then ligated in to yield the vectors pCGNN-1FRAPe-p65(450-550),pCGNN-3FRAPe-p65(450-550), pCGNN-1FRAPe-p65(361-550) andpCGNN-3FRAPe-p65(361-550). All constructs were verified by restrictionanalysis.

[0390] Vectors were also constructed that encode C-terminal fusions ofFRB domain(s) with portions of the p65 activation domain. PlasmidspCGNN-p65(450-550) and pCGNN-p65(361-550) were digested with SpeI andBamHI, and XbaI-BamHI fragments encoding 1 and 3 copies of FRAPe(isolated from pCGNN-GAL4-1FRAPe and pCGNN-GAIA-3FRAPe) and 1 copy ofFRB (isolated from pCGNN-GAL4-1FRB) ligated in to yield the plasmidspCGNN-p65(450-550)-1FRAPe, pCGNN-p65(450-550)-3FRAPe,pCGNN-p65(361-550)-1FRAPe, pCGNN-p65(361-550)-3FRAPe,pCGNN-p65(450-550)-1FRB and pCGNN-p65(361-550)-1FRB. All constructs wereverified by restriction analysis.

[0391] (e) Further Constructs

[0392] Other constructs can be made analogously with the aboveprocedures, but using alternative portions of the FRAP sequence or FRBdomains containing modified peptide sequence. For example, primers 12and 13 are used to amplify the entire coding region of FRAP. Primers 1and 13, 6 and 13, and 5 and 13, are used to amplify three fragmentsencompassing the FRB domain and extending through to the C-terminal endof the protein (including the lipid kinase homology domain). Thesefragments differ by encoding different portions of the proteinN-terminal to the FRB domain. In each case, RT-PCR is used as describedabove to amplify the regions from human thymus RNA, the PCR products arepurified, digested with XbaI and SpeI, ligated into XbaI-SpeI digestedpCGNN, and verified by restriction analysis and DNA sequencing.

[0393] (f) Primer Sequences 1 5′GCATGTCTAGAGAGATGTGGCATGAAGGCCTGGAAG 25′GCATCACTAGTCTTTGAGATTCGTCGGAACACATG 35′GCACATTCTAGAATTGATACGCCCAGACCCTTG 45′CGATCAACTAGTAAGTGTCAATTTCCGGGGCCT 55′GCACTATCTAGACTGAAGAACATGTGTGAGCACAGC 65′GCACTATCTAGAGTGAGCGAGGAGCTGATCCGAGTG 75′CGATCAACTAGTGGAAACATAAAGCAGCTCTAAGGA 85′CGATCAACfAG1TGGCACAGCCAATTCAAGGTCCCG 95′ATGCTCTAGACITGGGGGCCTTGCTTGGCAAC 10 5′ATGCTCTAGAGATGAGTTTCCCACCATGGTG11 5′GCATGGATCCGCTCAACTAGTGGAGCTGATCTGACTCAG 125′ATGCTCTAGACLTGGAACCGGACCTGCCGCC 135′GCATCACTAGTCCAGAAAGGGCACCAGCCAATAT

[0394] Restriction sites are underlined (XbaI=TCTAGA, SpeI=ACGAGT,BamHI=GGATCC).

[0395] (g) DNA sequence of representative final construct:pCGNN-ZFHD1-1FRB encoding a 12CA5 epitope-SV40 NLS-ZFHD1-FRB fusion isshown in WO 96/41865.

[0396] (h) Bicistronic Constructs

[0397] The internal ribosome entry sequence (IRES) from theencephalomyocarditis virus was amplified by PCR from pWZL-Bleo. Theresulting fragment, which was cloned into pBS-SK+ (Stratagene), containsan XbaI site and a stop codon upstream of the IRES sequence anddownstream of it, an NcoI site encompassing the ATG followed by SpeI andBamHI sites. To facilitate cloning, the sequence around the initiatingATG of pCGNN-ZFHD1-3FKBP was mutated to an NcoI site and the XbaI sitewas mutated to a NheI site using oligonucleotides shown in WO 96/41865.An NcoI-BamHI fragment containing ZFHD1-3FKBP was then cloned downstreamof pBS-IRES to create pBS-IRES-ZFHD1-3FKBP. The XbaI-BamHI fragment fromthis plasmid was next cloned into SpeI/BamHI-cut pCGNN-1FRB-p65(361-550)to create pCGNN-1FRB-p65(361-550)—IRES-ZFHD1-3FKBP.

[0398] C. Retroviral Vectors for the Expression of Chimeric Proteins

[0399] Retroviral vectors used to express transcription factor fusionproteins from stably integrated, low copy genes were derived frompSRaMSVtkNeo (Muller et al., MCB 11:1785-92, 1991) and pSRaMSV(XbaI)(Sawyers et al., J. Exp. Med. 181:307-313, 1995). Unique BamHI sites inboth vectors were removed by digesting with BamHI, filling in withKlenow and religating to produce pSMTN2 and pSMTX2, respectively. pSMTN2expresses the Neo gene from an internal thymidine kinase promoter. AZeocin gene (Invitrogen) is cloned as a NheI fragment into a unique XbaIsite downstream of an internal thymidine kinase promoter in pSMTX2 toyield pSNTZ. This Zeocin fragment was generated by mutagenizing pZeo/SV(Invitrogen) using the following primers to introduce NheI sitesflanking the zeocin coding sequence. Primer 15′-GCCATGGTGGCTAGCCTATAGTGAG Primer 2 5′-GGCGGTGTTGGCTAGCGTCGGTCAG

[0400] pSMTN2 contains unique EcoRI and HindIII sites downstream of theLTR. To facilitate cloning of transcription factor fusion proteinssynthesized as XbaI-BamHI fragments the following sequence was insertedbetween the EcoRI and HindIII sites to create pSMTN3: 12CA5 epitope                       M   A   S   S   Y   P   Y   D   V   P   D5′ gaattccagaagcgcgt ATG GCT TCT AGC TAT CCT TAT GAC GTG CCT GAC   EcoRI                                 SV40 T NLS Y   A   S   L   G   G   P   S   S   P   K   K   K   R   K TAT GCC AGCCTG GGA GGA CCT TCT AGTCCT AAG AAG AAG AGA AAG  V GTG TCT AGATAT CGA GGATCCCAA GCT T     XbaI             BamHI    HindIII

[0401] The equivalent fragment is inserted into a unique EcoRI site ofpSMTZ to create pSMTZ3 with the only difference being that the 3′HindIII site is replaced by an EcoRI site. pSMTN3 and pSMTZ3 permitchimeric transcription factors to be cloned downstream of the 5′ viralLTR as XbaI-BamHI fragments and allow selection for stable integrants byvirtue of their ability to confer resistance to the antibiotics G418 orZeocin respectively.

[0402] To generate the retroviral vector SMTN-ZFHD1-3FKBP,pCGNN-ZFHD1-3FKBP was first mutated to add an EcoRI site upstream of thefirst amino acid of the fusion protein. An EcoRI-BamHI(blunted) fragmentwas then cloned into EcoRI-HindIII(blunted) pSRaMSVtkNeo so thatZFHD1-3FKBP was expressed from the retroviral LTR.

Example 8 Rapamycin-Dependent Transcriptional Activation

[0403] In preliminary experiments, three copies of FKBP fused either toa Gal4 DNA binding domain or a transcription activation domain activatedboth the stably integrated or transiently transfected reporter gene morestrongly than corresponding fusion proteins containing only one or twoFKBP domains. To evaluate this parameter with FRB fusion proteins,effector plasmids containing Gal4 DNA binding domain fused to one ormore copies of an FRB domain were co-transfected with a plasmid encodingthree FKBP domains and a p65 activation domain (3×FKBP-p65) by transienttransfection. It was found that in this system, four copies of the FRBdomain fused to the Gal4 DNA binding domain activated the stablyintegrated reporter gene more strongly than other corresponding fusionproteins with fewer FRB domains.

[0404] Method: HT1080 B cells were grown in MEM supplemented with 10%Bovine Calf Serum. Approximately 4×10⁵ cells/well in a 6 well plate(Falcon) were transiently transfected by Lipofection procedure asrecommended by the supplier (GIBCO, BRL). The DNA: Lipofectamine ratioused in this experiment correspond to 1:6. Cells in each well recieved500 ng of pCGNN F3-p65, 1.9 ug of PUC 118 plasmid as carrier and 100 ngof one of the following plasmids: pCGNN Gal4-1FRB, pCGNN Gal4-2FRB,pCGNN Gal4-3FRB or pCGNN Gal4-4FRB. Following transfection, 2 ml freshmedia was added and supplemented with Rapamycin to the indicatedconcentration. After 24 hrs, 100 ul of the media was assayed for SEAPactivity as described (Spencer et al, 1993).

[0405] To test whether multiple FRB domains fused to a p65 activationdomain results in increased transcriptional activation of the reportergene, we co-transfected HT1080 B cells with plasmids expressingGal4-3×FKBP and 1, 2, 3 or 4 copies of FRB fused to p65 activationdomain. Surprisingly, unlike the DNA binding domain-FRB fusions, asingle copy of FRB fused to p65 activation domain activated the reportergene significantly more strongly than corresponding fusion proteinscontaining 2 or more copies of FRB.

[0406] Method: HT1080 B cells were grown in MEM supplemented with 10%Bovine Calf Serum. Approximately 4×10⁵ cells/well in a 6 well plate weretransiently transfected by Lipofection procedure as recommended byGIBCO, BRL. The DNA: Lipofectamine ratio used correspond to 1:6. Cellsin each well recieved 1.9 ug of PUC 118 plasmid as carrier, 100 ng ofpCGNNGal4F3 and 500 ng one of the following plasmids:pCGNN1, 2, 3 or 4FRB-p65. Following transfection, 2 ml fresh media was added andsupplemented with Rapamycin to the indicated concentration. After 24hrs, 100 ul of the media was assayed for SEAP activity as described(Spencer et al, 1993).

[0407] Similar experiments were also conducted using another stable cellline (HT1080 B14) containing the 5×Gal4-IL2-SEAP reporter gene and DNAsequences encoding a fusion protein containing a Gal4 DNA binding domainand 3 copies of FKBP stably integrated. These cells were transientlytransfected with effector plasmids expressing p65 activation domainfused to 1 or more copies of an FRB domain. Similar to our observationswith HT1080 B cells, in these experiments effector plasmids expressing asingle copy of FRB-p65 activation domain fusion protein activated thereporter gene more strongly than others with 2 or more copies of FRB.

Example 9

[0408] A. Rapamycin-Dependent Transcriptional Activation in TransientlyTransfected Cells: ZFHD1 and p65 Fusions

[0409] Human fibrosarcoma cells transiently transfected with a SEAPtarget gene and plasmids encoding representative ZFHD-FKBP— andFRB-p65-containing fusion proteins exhibited rapamycin-dependent anddose-responsive secretion of SEAP into the cell culture medium. See WO96/41865, FIG. 4A. SEAP production was not detected in cells in whichone or both of the transcription factor fusion plasmids was omitted, norwas it detected in the absence of added rapamycin (WO 96/41865, FIG.4B). When all components were present, however, SEAP secretion wasdetectable at rapamycin concentrations as low as 0.5 nM (WO 96/41865,FIG. 4A). Peak SEAP secretion was observed at 5 nM. Similar results havebeen obtained when the same transcription factors were used to driverapamycin-dependent activation of an hGH reporter gene or a stablyintegrated version of the SEAP reporter gene made by infection with aretroviral vector. It is difficult to determine the fold activation inresponse to rapamycin since levels of SEAP secretion in the absence ofdrug are undetectable, but it is clear that in this system there is atleast a 1000-fold enhancement over background levels in the absence ofrapamycin. Thus, this system exhibits undetectable background activityand high dynamic range.

[0410] Several different configurations for transcription factor fusionproteins were explored (See See WO 96/41865, FIG. 5). When FKBP domainswere fused to ZFHD1 and FRBs to p65, optimal levels of rapamycin-inducedactivation ocurred when there were multiple FKBPs fused to ZFHD1 andfewer FRBs fused to p65. The preference for multiple drug-bindingdomains on the DNA-binding protein may reflect the capacity of theseproteins to recruit multiple activation domains and therefore to elicithigher levels of promoter activity. The presence of only 1 drug-bindingdomain on the activation domain should allow each FKBP on ZFHD torecruit one p65. Any increase in the number of FRBs on p65 wouldincrease the chance that fewer activation domains would be recruited toZFHD, each one linked my multiple FRB-FKBP interactions.

[0411] Methods:

[0412] HT1080 cells (ATCC CCL-121), derived from a human fibrosarcoma,were grown in MEM supplemented with non-essential amino acids and 10%Fetal Bovine Serum. Cells plated in 24-well dishes (Falcon, 6×10⁴cells/well) were transfected using Lipofectamine under conditionsrecommended by the manufacturer (GIBCO/BRL). A total of 300 ng of thefollowing DNA was transfected into each well: 100 ng ZFHDx12-CMV-SEAPreporter gene, 2.5 ng pCGNN-ZFHD1-3FKBP or other DNA binding domainfusion, 5 ng pCGNN-1FRB-p65(361-550) or other activation domain fusionand 192.5 ng pUC118. In cases where the DNA binding domain or activationdomain were omitted an equivalent amount of empty pCGNN expressionvector was substituted. Following lipofection (for 5 hours) 500 μlmedium containing the indicated amounts of rapamycin was added to eachwell. After 24 hours, medium was removed and assayed for SEAP activityas described (Spencer et al, Science 262:1019-24, 1993) using aLuminescence Spectrometer (Perkin Elmer) at 350 nm excitation and 450 nmemission. Background SEAP activity, measured from mock-transfectedcells, was subtracted from each value.

[0413] To prepare transiently transfected HT1080 cells for injectioninto mice (See below), cells in 100 mm dishes (2×10⁶ cells/dish) weretransfected by calcium phosphate precipitation for 16 hours (Gatz, C.,Kaiser, A. & Wendenburg, R., 1991,Mol. Gen. Genet. 227, 229-237) withthe following DNAs: 10 mg of ZHWTx12-CMV-hGH, 1 mg pCGNN-ZFHD1-3FKBP, 2mg pCGNN-1FRB-p65(361-550) and 7 mg pUC118. Transfected cells wererinsed 2 times with phosphate buffered saline (PBS) and given freshmedium for 5 hours. To harvest for injection, cells were removed fromthe dish in Hepes Buffered Saline Solution containing 10 mM EDTA, washedwith PBS/0.1% BSA/0.1% glucose and resuspended in the same at aconcentration of 2×10⁷ cells/ml.

[0414] Plasmids: Construction of the transcription factor fusionplasmids is described above.

[0415] pZHWTx12-CMV-SEAP

[0416] This reporter gene, containing 12 tandem copies of a ZFHD1binding site (Pomerantz et al., 1995) and a basal promoter from theimmediate early gene of human cytomegalovirus (Boshart et. al., 1985)driving expression of a gene encoding secreted alkaline phosphatase(SEAP), was prepared by replacing the NheI-HindIII fragment of pSEAPPromoter (Clontech) with an NheI-XbaI fragment containing 12 ZFHDbinding sites shown in WO 96/41865 and an XbaI-HindIII fragmentcontaining a minimal CMV promoter (−54 to +45), also shown in WO96/41865.

[0417] pZHWTx12-CMV-hGH

[0418] Activation of this reporter gene leads to the production of hGH.It was constructed by replacing the HindIII-BamHI (blunted) fragment ofpZHWTx12-CMV-SEAP (containing the SEAP coding sequence) with a HindIII(blunted)-EcoRI fragment from pOGH (containing an hGH genomic clone;Selden et al., MCB 6:3171-3179, 1986; the BamHI and EcoRI sites wereblunted together).

[0419] pZHWTx12-IL2-SEAP

[0420] This reporter gene is identical to pZHWTx12-CMV-SEAP except theXbaI-HindIII fragment containing the minimal CMV promoter was replacedwith the following XbaI-HindIII fragment containing a minimal IL2 genepromoter (−72 to +45 with respect to the start site; Siebenlist et al.,MCB 6:3042-3049, 1986) (see WO 96/41865)

[0421] pLH

[0422] To facilitate the stable integration of a single, or few, copiesof reporter gene the following retroviral vector was constructed. pLH(LTR-hph), which contains the hygromycin B resistance gene driven by theMoloney murine leukemia virus LTR and a unique internal ClaI site, wasconstructed as follows: The hph gene was cloned as a HindIII-ClaIfragment from pBabe Hygro (Morganstern and Land, NAR 18:3587-96, 1990)into BamHI-ClaI cut pBabe Bleo (resulting in the loss of the bleo gene;the BamHI and HindIII sites were blunted together).

[0423] pLH-ZHWTx12-IL2-SEAP

[0424] To clone a copy of the reporter gene containing 12 tandem copiesof the ZFHD1 binding site and a basal promoter from the IL2 gene drivingexpression of the SEAP gene into the pLH retroviral vector, theMluI-ClaI fragment from pZHWTx12-IL2-SEAP (with ClaI linkers added) wascloned into the ClaI site of pLH. It was oriented such that thedirections of transcription from the viral LTR and the internal ZFHD-IL2promoters were the same.

[0425] pLH-G5-IL2-SEAP

[0426] To construct a retroviral vector containing 5 Gal4 sites embeddedin a minimal IL2 promoter driving expression of the SEAP gene, aClaI-BstBI fragment consisting of the following was inserted into theClaI site of pLH such that the directions of transcription from theviral LTR and the internal Gal4-IL2 promoters were the same: AClaI-HindIII fragment containing 5 Gal4 sites and regions −324 to −294and −72 to +45 of the IL2 gene (shown in WO 96/41865) and aHindIII-BstBI fragment containing the SEAP gene coding sequence (Bergeret al., Gene 66:1-10, 1988) mutagenized to add a BstB1 site immediatelyafter the stop codon (shown in WO 96/41865).

[0427] B. Rapamycin-Dependent Transcriptional Activation in StablyTransfected Cells

[0428] The following experiments confirmed that this system exhibitssimilar properties in stably transfected cells. We generated stable celllines by sequential transfection of a SEAP target gene and expressionvectors for ZFHD1-3FKBP and 1FRB-p65, respectively. A pool of severaldozen stable clones resulting from the final transfection exhibitedrapamycin-dependent SEAP production. From this pool, we characterizedseveral individual clones, many of which produced high levels of SEAP inresponse to rapamycin. Results from one such clone are shown in FIG. 4Cof WO 96/41865. This clone produced SEAP at levels approximately fortytimes higher than the pool and significantly higher than transientlytransfected cells. In an attempt to rigorously quantitate backgroundSEAP production and induction ratio in this clone, we performed a secondset of assays in which the length of the SEAP assay was increased by afactor of approximately 50 to detect any SEAP activity in untreatedcells. Under these conditions, mock transfected cells produced 47arbitrary fluorescence units, while the transfected clone produced 54units in the absence of rapamycin and over 90,000 units at 100 nMrapamycin. Thus, in this stable cell line, background gene expressionwas negligible and the induction ratio (7 units to 90,000 units) wasgreater than four orders of magnitude.

[0429] To simplify the task of stable transfection, we used abicistronic expression vector that directs the production of bothZFHD1-3FKBP and 1FRB-p65 through the use of an internal ribosome entrysequence (IRES). This expression plasmid was cotransfected, togetherwith a zeocin-resistance marker plasmid, into a cell line carrying aretrovirally-transduced SEAP reporter gene, and a pool of approximatelyfifty drug-resistant clones was selected and expanded. This pool ofclones also exhibited rapamycin-dependent SEAP production with nodetectable background and a very similar dose-response curve to thatobserved in transiently transfected cells. This pool would be expectedto contain individual clones with performance similar to the clonestudied in FIG. 4C of WO 96/41865. Thus, rapamycin-responsive geneexpression can be readily obtained in both transiently and stablytransfected cells. In both cases, regulation is characterized by verylow background and high induction ratios.

[0430] Stable cell lines. Helper-free retroviruses containing thereporter gene or DNA binding domain fusion were generated by transientco-transfection of 293T cells (Pear, W. S., Nolan, G. P., Scott, M. L. &Baltimore D., 1993, Production of high-titer helper-free retroviruses bytransient transfection. Proc. Natl. Acad. Sci. USA 90, 8392-8396) with aPsi(−) amphotropic packaging vector and the retroviral vectorspLH-ZHWTx12-IL2-SEAP or SMTN-ZFHD1-3FKBP, respectively. To generate aclonal cell line containing the reporter gene stably integrated, HT1080cells infected with retroviral stock were diluted and selected in thepresence of 300 mg/ml Hygromycin B. Individual clones from this andother cell lines described below were screened by transient transfectionof the missing components followed by the addition of rapamycin asdescribed above. All 12 clones analyzed were inducible and had little orno basal activity. The most responsive clone, HT1080L, was selected forfurther study.

[0431] HT20-6 cells, which contain the pLH-ZHWTx12-IL2-SEAP reportergene, ZFHD1-3FKBP DNA binding domain and 1FRB-p65(361-550) activationdomain stably integrated, were generated by first infecting HT1080Lcells with SMTN-ZFHD1-3FKBP-packaged retrovirus and selecting in mediumcontaining 500 mg/ml G418. A strongly responsive clone, HT1080L3, wasthen transfected with linearized pCGNN-1FRB-p65(361-550) and pZeoSV(Invitrogen) and selected in medium containing 250 mg/ml Zeocin.Individual clones were first tested for the presence of1FRB-p65(361-550) by western. Eight positive clones were analyzed byaddition of rapamycin. All eight had low basal activity and in six ofthem, gene expression was induced by at least two orders of magnitude.The clone that gave the strongest response, HT20-6, was selected forfurther analysis.

[0432] HT23 cells were generated by co-transfecting HT1080L cells withlinearized pCGNN-1FRB-p65(361-550)—IRES-ZFHD1-3FKBP and pZeoSV andselecting in medium containing 250 mg/ml Zeocin. Approximately 50 cloneswere pooled for analysis.

[0433] For analysis, cells were plated in 96-well dishes (1.5×10⁴cells/well) and 200 μl medium containing the indicated amounts ofrapamycin (or vehicle) was added to each well. After 18 hours, mediumwas removed and assayed for SEAP activity. In some cases, medium wasdiluted before analysis and relative SEAP units obtained multiplied bythe fold-dilution. Background SEAP activity, measured from untransfectedHT1080 cells, was subtracted from each value.

Example 10 Rapamycin-Dependent Production of hGH in Mice

[0434] In Vivo Methods: Animals, husbandry, and general procedures. Malenu/nu mice were obtained from Charles River Laboratories (Wilmington,Mass.) and allowed to acclimate for five days prior to experimentation.They were housed under sterile conditions, were allowed free access tosterile food and sterile water throughout the entire experiment, andwere handled with sterile techniques throughout. No immunocompromisedanimal demonstrated outward infection or appeared ill as a result ofhousing, husbandry techniques, or experimental techniques.

[0435] To transplant transiently transfected cells into mice, 2×10⁶transfected HT1080 cells, were suspended in 100 ml PBS/0.1% BSA/0.1%glucose buffer, and administered into four intramuscular sites(approximately 25 ml per site) on the haunches and flanks of theanimals. Control mice received equivalent volume injections of bufferalone.

[0436] Rapamycin was formulated for in vivo administration bydissolution in equal parts of N,N-dimethylacetamide and a 9:1 (v:v)mixture of polyethylene glycol (average molecular weight of 400) andpolyoxyethylene sorbitan monooleate. Concentrations of rapamycin, in thecompleted formulation, were sufficient to allow for in vivoadministration of the appropriate dose in a 2.0 ml/kg injection volume.The accuracy of the dosing solutions was confirmed by HPLC analysisprior to intravenous administration into the tail veins. Some controlmice, bearing no transfected HT1080 cells, received 10.0 mg/kgrapamycin. In addition, other control mice, bearing transfected cells,received only the rapamycin vehicle.

[0437] Blood was collected by either anesthetizing or sacrificing micevia CO2 inhalation. Anesthetized mice were used to collect 100 ml ofblood by cardiac puncture. The mice were revived and allowed to recoverfor subsequent blood collections. Sacrificed mice were immediatelyexsanguinated. Blood samples were allowed to clot for 24 hours, at 4°C., and sera were collected following centrifugation at 1000×g for 15minutes. Serum hGH was measured by the Boehringer Mannheim non-isotopicsandwich ELISA (Cat No. 1 585 878). The assay had a lower detectionlimit of 0.0125 ng/ml and a dynamic range that extended to 0.4 ng/ml.Recommended assay instructions were followed. Absorbance was read at 405nm with a 490 nm reference wavelength on a Molecular Devices microtiterplate reader. The antibody reagents in the ELISA demonstrate no crossreactivity with endogenous, murine hGH in diluent sera or nativesamples.

[0438] hGH expression In Vivo. For the assessment of dose-dependentrapamycin-induced stimulation of hGH expression, rapamycin wasadministered to mice approximately one hour following injection ofHT1080 cells. Rapamycin doses were either 0.01, 0.03, 0.1, 0.3, 1.0,3.0, or 10.0 mg/kg. Seventeen hours following rapamycin administration,the mice were sacrificed for blood collection.

[0439] To address the time course of in vivo hGH expression, micereceived 10.0 mg/kg of rapamycin one hour following injection of thecells. Mice were sacrificed at 4, 8, 17, 24, and 42 hours followingrapamycin administration.

[0440] The ability of rapamycin to induce sustained expression of hGHfrom transplanted HT1080 cells was tested by repeatedly administeringrapamycin. Mice were administered transfected HT1080 cells as describedabove. Approximately one hour following injection of the cells, micereceived the first of five intravenous 10.0 mg/kg doses of rapamycin.The four remaining doses were given under anesthesia, immediatelysubsequent to blood collection, at 16, 32, 48, and 64 hours. Additionalblood collections were also performed at 72, 80, 88, and 96 hoursfollowing the first rapamycin dose. Control mice were administeredcells, but received only vehicle at the various times of administrationof rapamycin. Experimental animals and their control counterparts wereeach assigned to one of two groups. Each of the two experimental groupsand two control groups received identical drug or vehicle treatments,respectively. The groups differed in that blood collection times werealternated between the two groups to reduce the frequency of bloodcollection for each animal.

[0441] Results

[0442] Rapamycin elicited dose-responsive production of hGH in theseanimals (FIG. 6 of WO 96/41865). hGH concentrations in therapamycin-treated animals compared favorably with normal circulatinglevels in humans (0.2-0.3 ng/ml). No plateau in hGH production wasobserved in these experiments, suggesting that the maximal capacity ofthe transfected cells for hGH production was not reached. Controlanimals-those that received transfected cells but no rapamycin and thosethat received rapamycin but no cells-exhibited no detectable serum hGH.Thus, the production of hGH in these animals was absolutely dependentupon the presence of both engineered cells and rapamycin.

[0443] The presence of significant levels of hGH in the serum 17 hoursafter rapamycin administration was noteworthy, because hGH is clearedfrom the circulation with a half-life of less than four minutes in theseanimals. This observation suggested that the engineered cells continuedto secrete hGH for many hours following rapamycin treatment. To examinethe kinetics of rapamycin control of hGH production, we treated animalswith a single dose of rapamycin and then measured hGH levels atdifferent times thereafter. Serum hGH was observed within four hours ofrapamycin treatment, peaked at eight hours (at over one hundred timesthe sensitivity limit of the hGH ELISA), and remained detectable 42hours after treatment. hGH concentration decayed from its peak with ahalf-life of approximately 11 hours. This half-life is severalhundredfold longer than the half-life of hGH itself and approximatelytwice the half-life of rapamycin (4.6 hr) in these animals. The slowerdecay of serum hGH relative to rapamycin could reflect the presence ofhigher tissue concentrations of rapamycin in the vicinity of theimplanted cells. Alternatively, persistence of hGH production from theengineered cells may be enhanced by the stability of hGH mRNA.

[0444] Interestingly, administration of a second dose of rapamycin tothese animals at 42 hr resulted in a second peak of serum hGH, whichdecayed with similar kinetics indicating that the engineered cellsretained the ability to respond to rapamycin for at least two days.Therefore, to ascertain the ability of this system to elevate andmaintain circulating hGH concentrations, we performed an experiment inwhich animals received multiple doses of rapamycin at 16-hour intervals.This interval corresponds to the time required for hGH levels to peakand then decline approximately half-way. According to this regimen,rapamycin concentration is predicted to approach a steady-state troughconcentration of 1.7 μg/ml after two doses (shown as dotted line in FIG.8 of WO 96/41865). hGH levels should also approach a steady state troughconcentration following the second dose. Treated animals indeed heldrelatively stable levels of circulating hGH in response to repeateddoses of rapamycin. After the final dose, hGH levels remained constantfor 16 hours and then declined with a similar half-life as rapamycin(6.8 hours for hGH versus 4.6 hours for rapamycin). These data suggestthat upon multiple dosing, circulating rapamycin imparts tight controlover the secretion of hGH from transfected cells in vivo. In particular,it is apparent that protein production is rapidly terminated uponwithdrawal of drug.

[0445] Discussion

[0446] These experiments demonstrate the feasibility of controlling theproduction of a secreted therapeutic protein from genetically engineeredcells using a small-molecule drug. This system has many of the featuresrequired for use in human gene and cell therapy. It is characterized byvery low background activity and high induction ratio. It functionsindependently of host physiology or any cell-type-specific factors. Itis composed completely of human proteins. The controlling drug is wellbehaved in vivo and orally bioavailable.

[0447] With a system of this general design, it should be possible toprovide stable and precisely titrated doses of secreted therapeuticproteins from engineered cells in vivo. Intermittent and pulsatiledosing should also be feasible. A considerable advantage of proteindelivery from engineered cells under small-molecule control is that therate of protein production at any given time is a function of thecirculating concentration of the small-molecule drug. Therefore, theapparent pharmacokinetics of a therapeutic protein such as hGH can bedramatically altered. In our experiments, for example, the kinetics ofcirculating hGH delivered from engineered cells following a singleadministration of rapamycin are markedly different from those observedfollowing a single administration of recombinant protein. hGHadministered to mice intravenously is cleared with a half-time of a fewminutes, whereas hGH levels from engineered cells induced with rapamycindecayed with a half-time of approximately eleven hours. Even in humans,where the half-time for hGH clearance is approximately twenty minutes,injections must be given every other day, and serum hGH levels fluctuatedramatically. It is likely that protein delivery from engineered cellsunder precise pharmacologic control will lead to more effective therapy,particularly for proteins with poor pharmacokinetics or low therapeuticindex.

[0448] The use of a small-molecule drug to link a DNA-binding domain andactivation domain is an effective strategy for regulating geneexpression in vivo. One especially attractive feature is that the systemis entirely modular, allowing each component to be optimized andengineered independently. In contrast to bacterial repressors, whichrely on relatively subtle allosteric intramolecular interactions tocontrol DNA-binding activity, the dimerization strategy can be adaptedto virtually any DNA-binding and activation domain. We have used here aDNA-binding domain of defined structure which readily supports rationalengineering of DNA-binding affinity and new recognition specificities.Similarly, activation domains can be engineered for maximal potency andother suitable properties. Indeed, the engineered transcription factorsused in these experiments elicit very high levels of gene expressionrelative to conventional promoter/enhancer systems, and furtherenhancements in either domain can be readily incorporated. The abilityto introduce engineered transcription factors dedicated to thetranscription of a single target gene provides opportunities to achievelower backgrounds and substantially higher levels of gene expression invivo than conventional expression vectors.

[0449] We have also chosen to construct our regulated transcriptionfactors from human proteins to minimize the potential for recognition bythe immune system. It has been reported that autologous T cellsexpressing a fusion protein composed of bacterial hygromycinphosphotransferase and herpes virus thymidine kinase were effectivelyrecognized and eliminated by host cytotoxic T cells, even in AIDSpatients with debilitated immune systems (Riddell, S. R., et al. T-cellmediated rejection of gene-modified HIV-specific cytotoxic T lymphocytesin HIV-infected patients. Nature Med. 2, 216-223 (1996)). Thisobservation suggests that the risk of immune recognition of heterologousproteins in engineered cells is a real one and that, therefore, the useof human proteins for performing regulatory functions in human cells isprudent. Although each individual component of our transcription factorfusion proteins is human in sequence, each protein contains junctionpeptides which could potentially be recognized as foreign. Thesejunctions may be designed or selected, however, to minimize theirpresentation to the immune system, as discussed previously.

[0450] The principal limitation of rapamycin-based systems is the nativebiological activity of rapamycin, which, through inhibition of FRAPactivity blocks cell-cycle progression leading to immunosuppression invivo. However, the ability to introduce substituents or otherwise modifythe structure of rapamycin to substantially reduce or abolish binding toFKBP and/or FRAP provides access to rapalogs devoid of undueimmunosuppressive activity. Use of such rapalogs, especially improvedrapalogs of this invention, together with correspondingly engineeredFKBP and/or FRB domains should prove widely useful for the regulation ofengineered protein production as well as the regulation of a widevariety of other biological processes in experimental animals and humangene therapy.

Example 11 FP Assay for Rapalog Binding to FKBP

[0451] Affinities of rapalogs for FKBP proteins may be determined usinga competitive assay based on fluorescence polarization (FP). Afluorescein-labelled FK506 probe (AP1491) was synthesized as describedin WO 96/41865 (See Example 6 therein), and the increase in thepolarization of its fluorescence used as a direct readout of % boundprobe in an equilibrium binding experiment containing sub-saturatingFKBP and variable amounts of rapamycin analog as competitor.

[0452] Determination of Binding Affinities (IC50s) of Rapalogs Using FP

[0453] Serial 10-fold dilutions of each analog are prepared in 100%ethanol in glass vials and stored on ice. All other manipulations areperformed at room temperature. A stock of recombinant pure FKBP(purified by standard methods, see eg. Wiederrecht, G. et al. 1992. J.Biol. Chem. 267, 21753-21760) is diluted to 11.25 nM in 50 mM potassiumphosphate pH 7.8/150 mM NaCl/100 μg/ml bovine gamma globulin (“FPbuffer”: prepared using only low-fluorescence reagents from Panvera) and98 μl aliquots transferred to wells of a Dynatech micro-fluor black96-well fluorescence plate. 2.0 μl samples of the rapalogs are thentransferred in duplicate to the wells with mixing. Finally, a probesolution is prepared containing 10 nM AP1491 in 0.1% ethanol/FP buffer,and 100 μl added to each well with mixing. Duplicate control wellscontain ethanol instead of rapalog (for 100% probe binding) or ethanolinstead of rapalog and FP buffer instead of FKBP (0% binding).

[0454] The plates are stored covered in the dark for approximately 30min to permit equilibration and then the fluorescence polarization ofthe sample in each well is read on a Jolley FPM-2 FP plate reader(Jolley Consulting and Research, Inc., Grayslake, Ill.) in accordancewith the manufacturer's recommendations. The mean polarization. (mPunits) for each competitor concentration is usually converted to % totalbinding by reference to the control values and plotted (y) vs. log molarfinal concentration of competitor (x). Non-linear least square analysiswas used to fit the curve and extract the IC50 using the followingequation:

y=M1+(M4−M1)/(1+exp(M2*(M3−x)))

[0455] where M3 is the IC50. For incomplete curves the IC50 isdetermined by interpolation. Rapamycin and C14-desoxo-rapamycin may beincluded as controls in each case (C14-desoxo-rapamycin was prepared asdescribed by Luengo, J. I. et al. 1994 Tetrahedron Lett. 35, 6469-6472).

Example 12 Rapalog-Dependent Transcriptional Activation in TransientlyTransfected Cells

[0456] Rapalogs may be assayed for their ability to dimerize FKBP andFRB fusion proteins using a transcription read out as follows.Constructs encoding rapalog-dependent transcription factor fusionproteins are introduced into cells which contain, or are engineered tocontain, a reporter gene linked to transcriptional regulatory DNApermitting reporter gene expression following rapalog-dependentdimerization of the transcription factor fusion proteins. Thetranscription factor fusion proteins include (a) an FKBP fusion proteincontaining, as a heterologous effector domain, a DNA binding domain(DBD) and (b) an FRB fusion protein containing, as a heterologouseffector domain, a transcription activator domain. The FKBP and/or FRBdomains may contain naturally occurring or non-naturally occurringpeptide sequence. The presence of a rapalog which is capable ofmediating dimerization of the two fusion proteins leads to expression ofthe reporter gene. The level of reporter observed is indicative of theactivity of the rapalog as a dimerizer. Use of a target gene of interestin place of a reporter gene renders this a regulated gene expressionsystem for use in cells grown in culture or in whole organisms.

[0457] We have made use of such as system as follows: HT1080 cells (ATCCCCL-121), derived from a human fibrosarcoma, were grown in MEMsupplemented with non-essential amino acids and 10% Fetal Bovine Serum.Cells plated in 24-well dishes (Falcon, 6×10⁴ cells/well) weretransfected using Lipofectamine under conditions recommended by themanufacturer (GIBCO/BRL). A total of 300 ng of the following DNA wastransfected into each well:

[0458] (a) 100 ng ZFHDx12-CMV-SEAP reporter gene (reporter gene linkedto 12 recognition sites for the ZFHD1 DNA-binding domain),

[0459] (b) 2.5 ng pCGNN-ZFHD1-3FKBP or other DNA binding domain fusion(fusion protein comprising 3 FKBP domains and one ZFHD1 domain),

[0460] (c) 5 ng pCGNN-1FRB-p65(361-550) (fusion protein comprising anFRB domain and and a p65 transcription activation domain) and

[0461] (d) 192.5 ng pUC118.

[0462] In some experiments, pCGNN-1FRB(T2098L)-p65(361-550) was used inplace of pCGNN-1FRB-p65(361-550) to generate an FRB fusion proteincontaining an engineered FRB domain. In control experiments where theDNA binding domain or activation domain were omitted, an equivalentamount of empty pCGNN expression vector was substituted. For detailedinformation on the assembly and use of constructs including thosementioned herein, see WO 96/41865 (Clackson et al), especially theExamples therein (which are specifically incorporated by referenceherein). Following lipofection (for 5 hours) 500 ul medium containingthe indicated amounts of rapalog was added to each well. After 24 hours,medium was removed and assayed for SEAP activity as described (Spenceret al, Science 262:1019-24, 1993). Human fibrosarcoma cells transientlytransfected with a SEAP target gene and plasmids encoding representativeZFHD-FKBP- and FRB-p65-containing fusion proteins exhibitedrapalog-dependent and dose-responsive secretion of SEAP into the cellculture medium. SEAP production was not detected in cells in which oneor both of the transcription factor fusion plasmids was omitted, nor wasit detected in the absence of added rapalog. As shown in FIG. 1, cellstransfected with wild-type FKBP and FRB constructs exhibited SEAPproduction at dimerizer concentrations as low as 1 nM. FIG. 2illustrates preferential stimulation of SEAP production in cellsexpressing a mutant FRB (T2098L, FIGS. 2B and 2D; T2098F, FIG. 2E) ascompared to wild-type (FIGS. 2A and 2C). Similar results have beenobtained when the same transcription factors were used to driverapalog-dependent activation of an hGH target gene or a stablyintegrated version of the SEAP reporter gene made by infection with aretroviral vector.

Example 13 Mutagenesis and Phage Display to Generate ModifiedLigand-Binding Domains Complementary to Various Rapalogs

[0463] A. Engineered FKBP and FRB Domains

[0464] We have designed and prepared recombinant DNA constructs encodingthe fusion proteins tabulated below which bear illustrative modifiedligand-binding domains. Except a otherwise stated, mutants weregenerated using oligonucleotide-mediated site-directed mutagenesisaccording to standard methods (Kunkel, T. A., Bebenek, K. and McClary,J. 1991. Meth Enzymol. 204, 235-139), and confirmed by dideoxysequencing. Fusion Proteins containing modified FKBP domains (F36VhFKBP12)---p65 (F36V hFKBP12)---(F36V hFKBP12)---p65 (F36VhFKBP12)---(F36V hFKBP12)---(F36V hFKBP12)---p65 (F36M hFKBP12)---p65(F36M hFKBP12)---(F36M hFKBP12)---p65 (F36M hFKBP12)---(F36MhFKBP12)---(F36M hFKBP12)---p65 (F36V hFKBP12)---ZFHD1 (F36VhFKBP12)---(F36V hFKBP12)---ZFHD1 (F36V hFKBP12)---(F36VhFKBP12)---(F36V hFKBP12)---ZFHD1 (F36M hFKBP12)---ZFHD1 (F36MhFKBP12)---(F36M hFKBP12)---ZFHD1 (F36M hFKBP12)---(F36MhFKBP12)---(F36M hFKBP12)---ZFHD1 myr-(F36V hFKBP12)---(F36VhFKBP12)---Fas myr-(F36M hFKBP12)---(F36M hFKBP12)---Fas myr-(F36AhFKBP12)---(F36A hFKBP12)---Fas myr-(F36S/F99A hFKBP12)---(F36S/F99AhFKBP12)---Fas

[0465] We have also prepared constructs encoding the following FRBfusion proteins: Fusion Proteins containing modified (hFRAP) FRB domains(T2098A FRB)---p65 (T2098N FRB)---p65 (D2102A FRB)---p65 (Y2038HFRB)---p65 (Y2038L FRB)---p65 (Y2038A FRB)---p65 (F2039H FRB)---p65(F2039L FRB)---p65 (F2039A FRB)---p65 (K2095S/D2096N/T2098N FRB)---p65(TOR2 FRB)---p65

[0466] Yeast and Candida FRBs, modified by analogy to the modified hFRAPFRB domains discussed herein, may also be prepared by substitution of acodon for a different amino acid in place of one or more of the twoconserved Phe residues and the conserved Asp and Asn residues withineach of their FRB domains. Illustrative modified FRB domains derivedfrom TOR 1 and TOR2, include the following: Modified TOR1 and TOR2 FRBDomains TOR1 TOR2 F1975H F1978H F1975L F1978L F1975A F1978A F1975SF1978S F1975V F1978V F1976H F1979H F1976L F1979L F1976A F1979A F1976SF1979S F1976V F1979V D2039A D2042A N2035A N2038A N2035S N2038S

[0467] B. Testing Rationally Designed FKBP Mutants for Binding toRapalogs

[0468] An expression vector based on pET20b (Novagen) was constructedusing standard procedures that expresses FKBP preceded by ahexahistidine tag and a portion of the H. influenza hemaglutinin proteinthat is an epitope for the monoclonal antibody 12CA5. The sequence ofthe protein encoded by this vector is as follows:

[0469] His6 HA tagFKBP->MHHHHHHYPYDVPDYAAMAHMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDV ELLKLE

[0470] To generate expression vectors for FKBPs mutated at rapamycincontact residues, oligonucleotide-mediated site-directed mutagenesis wasperformed on the single-stranded form of the vector prepared from E.coli CJ236, as described (Kunkel, T. A., Bebenek, K. and McClary, J.1991. Meth Enzymol. 204, 235-139). Mutants were confirmed by dideoxysequencing. Mutant proteins were expressed in E. coli BL21(DE3)(Novagen) as described (Wiederrecht, G. et al. 1992. J. Biol. Chem. 267,21753-21760), and purified to homogeneity as described (Cardenas, M. E.et al. 1994. EMBO J. 13, 5944-5957).

[0471] Using this protocol the following mutant human FKBP12 proteinswere generated, using the indicated oligonucleotide primers (mutatedbases in upper case; 5′->3′):

[0472] Mutants Designed for Binding to C24 Rapalogs: Phe46HisagcataaacttaTGgggcttgtttctg (1) Phe46Leu agcataaacttTaagggcttgtttctg (2)Phe46Ala agcataaacttaGCgggcttgtttctg (3) Phe48HisttgcctagcataTGcttaaagggcttg (4) Phe48Leu ttgcctagcatTaacttaaagggcttg (5)Phe48Ala ttgcctagcataGCcttaaagggcttg (6) Glu54AlacctcggatcaccGCctgcttgcctag (7) Val55Ala cagcctcggatcGCctcctgcttgcc (8)

[0473] Mutants Designed for Binding to C13/C14 Rapalogs: Phe36AlaccgggaggaatcGGCtttctttccatcttc  (9) Phe36ValccgggaggaatcGACtttctttccatcttc (10) Phe36SerccgggaggaatcAGAtttctttccatcttc (11) Phe36MetccgggaggaatcCATtttctttccatcttc (12) (Phe36Met + Phe99Ala)aagctccacatcGGCgacgagagtggc (13)+primer 12 (Phe36Met + Phe99Gly)aagctccacatcGCCgacgagagtggc (14)+primer 12 (Phe36Ala + Phe99Ala) primer9 + primer 13 (Phe36Ala + Phe99Gly) primer 9 + primer 14 Tyr26AlacaagcatcccggtgGCgtgcaccacgcag (15) Asp37AlatcccgggaggaaGCaaatttctttccatc (16)

[0474] Mutant Designed for Binding to C28/C30 Rapalogs:

[0475] Glu54Ala cctcggatcaccGCctgcttgcctag (17)

[0476] To assay the relative binding affinity of rapamycin and rapalogsto FKBP mutants, a competitive fluorescence polarization (FP) assay isused that relies on the retention of FK506 (and hence probe) bindingaffinity by the mutants. The procedure is identical to that described inExample 12 except that a direct binding assay is first performed todetermine the dilution (concentration) of mutant FKBP to use in thecompetition reactions in order to obtain sub-saturation. Serialdilutions of mutant FKBP are made in FP buffer (Example 12) in 100 μlvolumes in Dynatech micro-fluor plates, and then 100 μl of 10 nM AP1491(probe) in [FP buffer+2% ethanol] added to each well. Equilibration andplate reading are as in Example 12. A plot of mP units vs concentrationof FKBP mutant is fit to following equation:

y=M3+(((x+M1+M2)−SQRT(((x+M1+M2){circumflex over( )}2)−(4*x*M1)))/(2*(M1)))*(M4−M3)

[0477] and the final mutant concentration/dilution at which 90% of probeis specifically bound is determined by interpolation. This finalconcentration is then used in a competition FP assay carried out as inExample 12, with 2× the final concentration of mutant replacing 11.25 nMFKBP in the protocol. Instead of 90% saturation, 75% can be selected toimpart greater sensitivity to the competition assay. Serial dilutions ofrapamycin analogs are used as competitor and the results are expressedas IC50 for each rapalog binding to each mutant.

[0478] C. Testing Rationally Designed FRB Mutants for Binding toFKBP-rapalog Complexes

[0479] A NcoI-BamHI fragment encoding residues 2021-2113 (inclusive) ofhuman FRAP was generated by PCR with primers 28 and 29 (below), andcloned into a derivative of pET20b(+) (Novagen) in which the NdeI siteis mutated to NcoI, to create pET-FRAP(2021-2113). Single-stranded DNAof this vector was used as a template in site-directed mutagenesisprocedures, as described above, to generate vectors encoding FRAPsmutated at rapamycin contact residues. Mutants were confirmed by dideoxysequencing. Mutants were then amplified by PCR using primers (30 and 31)that append XbaI and SpeI sites, and cloned into XbaI-SpeI digestedpCGNN-FRB-p65(361-550) (Example 7) to generate a series of constructsdirecting mammalian expression of chimeric proteins of the formE-N-mutant FRAP(2021-2113)-p65(361-550), where E indicates HA epitopetag and N indicates nuclear localization sequence. Constructs wereverified by restriction digestion and dideoxy sequencing.

[0480] Using this procedure the following constructs encoding candidatemutant FRAPs for binding to C7 rapalogs, each fused to the p65(361-550)activation domain, were generated using the indicated oligonucleotideprimers (mutated bases in upper case; 5′->3′): Tyr2038HiscctttccccaaagtGcaaacgagatgc (18) Tyr2038Leu cctttccccaaagAGcaaacgagatgc(19) Tyr2038Ala cctttccccaaagGCcaaacgagatgc (20) Phe2038HisgttcctttccccAtGgtacaaacgagatg (21) Phe2038LeugttcctttccccTaagtacaaacgagatg (22) Phe2038AlagttcctttccccaGCgtacaaacgagatg (23) Thr2098AlagtcccaggcttggGCgaggtccttgac (24) (Lys2095Ser + Asp2096Asn + Thr2098Asn)gtcccaggcttggTTgaggTTcGAgacattccctgatttc (25) Thr2098AsngtcccaggcttggTTgaggtccttgac (26) Asp2102AlacatgataatagaggGCccaggcttgggtg (27)

[0481] To assay the relative binding affinity of these mutants forcomplexes of FKBP with rapamycin and various rapalogs, each construct istransiently co-transfected into human HT1080B14 cells, as described inExample 8. Following transfection, serial dilutions of rapamycin orrapalog are added to the culture medium. After 24 hours, SEAP activityis measured as described in Example 8; the potency of SEAP activation atvarious rapalog concentrations is proportional to the affinity of theFRAP mutant for the complex between FKBP and the rapalog.

[0482] PCR Primers (Restriction Sites Upper Case; 5′->3′):gcatcCCATGGcaatcctctggcatgagatgtggcatgaaggcctggaag (28)cgtgaGGATCCtactttgagattcgtcggaacac (29)gcatcTCTAGAatcctctggcatgagatgtggcatgaaggcctggaag (30)ggtctGGATCCctaataACTAGTctttgagattcgtcggaacacatg (31)

[0483] D. Functional Display of FKBP and FRB Domains on FilamentousBacteriophage: One Approach to Selection as an Alternative to RationalDesign of Modified Domains

[0484] A phage display system for the display and selection of mutantFKBP and FRB domains is disclosed in detail in WO 96/41865 (Clackson etal), including vector construction, preparation of His6-flag-FKBP,pCANTAB-AP-FKBP, Binding enrichments, Primers, the Sequence ofpCANTAB-AP-FRAP(2015-2114) and pCANTAB-AP-FKBP, the synthesis ofbiotinylated FK506 for affinity enrichment studies, functional FKBPdisplay by competitive ELISA using biotinylated FK506, generation of alibrary of mutant FKBPs on phage targetted to the C13 and C14 positionsof rapamycin andliibrary sorting.

Example 14 Rapamycin-Dependent Activation of Signal Transduction

[0485] Many cellular receptors can be activated by aggregation, eitherby their physiological ligand or by anti-receptor antibodies.Additionally, the aggregation of two different proteins can oftentrigger an intracellular signal. Rapamycin and its analogs may be usedto trigger activation of a receptor effector domain by oligomerizingchimeric proteins, one of which contains one or more FKBPs and aneffector domain and the other of which contains one or more FRAP domainsand an effector domain. This scheme is illustrated in Figure T1(a).While both proteins are shown anchored to the membrane, a single onecould be membrane anchored, and addition of rapamycin or analog wouldrecruit the second protein to the membrane via dimerization. Membraneanchoring may be effected through a transmembrane protein anchor orthrough lipid modification of the protein(s), such as myristoylation.The same effector domain may be present on both proteins, or differentprotein domains that interact functionally may be used, such as aprotein kinase and a protein kinase substrate. Alternatively, a secondeffector may serve to inhibit the activity of the first effector.

[0486] We note that in some embodiments, the chimeric proteins are mixedchimeras, discussed previously, and contain FKBP and FRAP domainstogether with the heterologous efector domain. Oligomerization of asingle mixed chimera may also be used to activate signal transduction,as shown in Figure T1(b). Here rapamycin is shown to dimerize twoidentical copies of the protein. Reiteration of the FKBP and FRAPdomains permits higher multiples to occur, subject to geometricconstraints.

[0487] Two examples of the use of rapamycin in signal transduction areto trigger receptor tyrosine kinase activation and to trigger apoptosisvia Fas activation, both of which are discussed below. Unless otherwisementioned all DNA manipulations were performed following standardprocedures (F. M. Ausubel et al., Eds., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 1994) and all protein protocolswere performed following standard procedures (Harlow, E. and Lane, D.1988. Antibodies, a Laboratory Manual. Cold Spring Harbor Laboratory,Cold Spring Harbor.). All PCR products used to make constructs wereconfirmed by sequencing.

[0488] A. Rapamycin-Inducible Receptor Tyrosine Kinase Activation.

[0489] 1. Construction of pCM, an Expression Vector Containing aMyristylation Signal.

[0490] A XbaI-Myr-BamHI cassette, obtained by annealing oligonucleotides1 and 2, was digested with XbaI/BamHI and cloned into the XbaI/BamHIsite of the pCG expression vector (Tanaka, M. and Herr, W. 1990. Cell60: 375-386) to create pCGM. (For oligonucleotide sequences, see (7)below). This oligonucleotide cassette consists of an inframe XbaI sitefollowed by sequence encoding for the first 15 amino acids residues ofc-Src tyrosine kinase that has been shown to allow myristoylation andtarget protein to the plasma membrane (Cross et al., 1984. MCB.4:1834-1842). The myristoylation domain is followed by an inframe SpeIsite and stop codons. The XbaI site in the pCG vector is placed suchthat it adds two amino acids between the initiating Met and the sequencecloned. Since the spacing between the initiating Met and themyristylated Gly is crucial for membrane localization of c-Src (Pellmanet al. 1985. PNAS. 82: 1623-1627) the XbaI site following the ATG inpCGM was deleted by site directed mutagenesis following manufacturersprotocol (Muta-Gene, BioRad). To facilitate future cloning steps theSpeI site in the myristylation cassette was mutated to a XbaI site.Single stranded uracil-DNA of pCGM was prepared and the mutagenesis wascarried out using both oligonucleotide 3 (to delete the XbaI sitefollowing ATG and add an EcoRI site 5′ to ATG) and oligonucleotide 4 (tochange the SpeI site following the myristylation domain to a XbaI site).The resulting sequence surrounding the ATG of the pCM vector wasconfirmed by sequencing using oligonucleotide 5 (see sequence 1, (8)below).

[0491] 2. Addition of FKBPs and an Epitope Tag to pCM GeneratespCMF1/2/3.HA.

[0492] A SpeI-HA-BamHI cassette was prepared by annealing complementaryoligonucleotides (oligonucleotides 6 and 7). This cassette has aninframe SpeI site followed by nine amino acids of H. influenzaehemaglutinin gene that is recognized by the monoclonal antibody 12CA5,stop codons and a BamHI site. The SpeI-HA-BamHI cassette was sub clonedinto the SpeI/BamHI site of pCGNNF1, pCGNNF2 and pCGNNF3. Subsequently,the 1/2/3 copies of FKBP fused with HA epitope was sub cloned as anXbaI/BamHI fragment into pCM. The resulting plasmid (pCMF1/2/3.HA) hasthe following features: myristylation domain; an inframe XbaI site;one/two/three copies of FKBP; an inframe SpeI site; a HA epitope tag;and stop codons.

[0493] 3. Addition of FRBs and an Epitope Tag to pCM GeneratespCMFR1/2/3.Flag.

[0494] A SpeI-Flag-BamHI cassette can be prepared by annealingcomplementary oligonucleotides (oligonucleotides 8 and 9). This cassettehas the same features as the SpeI-HA-BamHI cassette described above withthe exception that the inframe SpeI site is followed by sequence thatcodes for eight amino acids (DYKDDDDY) (Hopp et al., 1988. Biotech. 6:1205-1210) that is recognized by a monoclonal antibody anti-FLAG.M2(Kodak Scientific Imaging Systems). The SpeI-Flag-BamHI cassette is subcloned into the SpeI/BamHI site in pCGNN-1FRB, pCGNN-2FRB and,pCGNN-3FRB. Subsequently 1/2/3 copies of FRB domain-Flag epitope fusionsare sub cloned as a XbaI/BamHI fragment into pCM. The resulting plasmid(pCMFR1/2/3.Flag) has the following features: myristylation domain; aninframe XbaI site; one/two/three copies of FRB; an inframe SpeI site; aFlag epitope tag; and stop codons.

[0495] 4. Fusion of FKBP and FRB Constructs to Receptor Tyrosine KinaseCytoplasmic Domain

[0496] The cytoplasmic domain of receptor tyrosine kinase of choice(e.g., EGFR, erbB-2, PDGFR, KDR/Flk-1, Flt-1) is PCR amplified withinframe 5′XbaI and 3′ SpeI sites. The PCR product may be subclonedeither into the inframe XbaI site such that the XbaI site is restored,or into the inframe SpeI site such that the SpeI site is restored inpCMFR series or pCMFseries vectors (see above). As a result, theFKBP/FRB domain(s) can be placed either C-terminal or NH2-terminal tothe cytoplasmic domain of the receptor tyrosine kinase. The vectors areconstructed such that (i) the cytoplasmic domain of a given receptor isfused to both FKBP and FRB (for e.g., EGFR cytoplasmic domain fused toeither FKBP or FRB) or (ii) can be constructed such that cytoplasmicdomains of two different receptors are fused to FKBP and FRB (for e.g.,EGFR cytoplasmic domain fused to FKBP and erbB-2 cytoplasmic domainfused to FRB). In the former case (i) addition of the drug, rapamycin,will induce the formation of homodimers (e.g., EGFR/EGFR) while, in thelatter (ii) addition of the drug will induce heterodimer (e.g.,EGFR/erbB-2) and result in activation of the signal transductioncascade.

[0497] 5. Testing the Constructs

[0498] To test the ability of rapamycin or analog to induce dimerizationof FKBP- and FRB-receptor cytoplasmic domain fusions, the constructs ofchoice (e.g., pCMEGFR-FR1 and pCMEGFR-F1) are cotransfected into Cos-1cells by lipofecfion (Gibco BRL). Three days after transfection thecells are induced with rapamycin and lysed in lysis buffer (1% TritonX-100; 50 mM Tris.cl pH8.0; 150 mM NaCl; 5 mM NaF; 1 mM sodium orthovanadate; 10 ug/ml aprotinin; 10 ug/ml leupeptin). The fusion proteinsfrom rapamycin-treated and untreated cell lysates are immunoprecipitatedwith anti-Flag and 12CA5 antibodies and immunoblotted withanti-phosphotyrosine antibody. The choice of cell type; the amount ofDNA transfected; the concentration of rapamycin used and the duration ofdrug treatment are varied to achieve optimal results.

[0499] 6. Rapamycin-Inducible Cell Growth

[0500] A selected mammalian cell line (e.g., NIH3T3) is cotransfectedwith constructs encoding for FRB and FKBP fusion proteins (e.g.,pCMEGFR-FR1 and pCMEGFR-F1) and stable cell lines expressing the fusionproteins are established. To determine whether rapamycin-inducibleactivation of receptor cytoplasmic domain will induce cellproliferation, stable cell lines expressing the fusion proteins aregrown either in the presence or absence of rapamycin and the changes incell growth rate are determined by routine procedures (e.g., bymonitoring cell number; by determining the 3H thymidine incorporationrate, etc.). The choice of receptor tyrosine kinase; the type ofreceptor activation (homodimer vs. heterodimer) may be chosen to obtainoptimal results.

[0501] 7. Oligonucleotide Sequences 1:CATGTCTAGAGGGAGTAGCAAGAGCAAGCCTAAGGACCCCAGCCAG CGCACTAGTTAAGAATTCTGATGATCAGCGGATCCTAGC 2: GCTAGGATCCGCTGATCATCAGAATTCTTAACTAGTG   CGCTGGCTGGGGTCCTTAGGCTTGCTTTGCTACTCCCTCTAGACATG 3:CGCCTTGTAGAA1TCGCGCGTATGGCGAGTAGCAAGA 4: CCCAGCCAGCGCTCTAGATAAGAATTCTGA5: AAGGGTCCCCAAACTCAC 6: GCATGACTAGUATCCGTACGACGTACCAGACT     ACGCATAAGAAAAGTGAGGATCGTACGG 7: CCGTAGGATCCTCATTTTCTTATGCGTAGTCTGGT   ACGTCGTACGGATAACTAGTCATGC 8: CCGTAGGATCCTCACTTTTCTTAATAATCGTCATCG  TCTLTGTAGTCACTAGTCATGC 9: GCATGACTAGTGACTACAAAGACGATGACGATTA  TTAAGAAAAGTGAGGATCGTACGG 8. Sequence 1:                          M    G    S    S    K    S    K    P    K   CGC CTT GTA GAA ttc GCG CGT ATG ggg agt agc aag agc aag cct aag D   P   S   Q   R   S   R   stop            stop gac ccc agc cag cgctct aga taa gaa ttc tga tga tca gcG GAT CCT GAG AAC T

[0502] The modified sequences are in lowercase bold and the intitiatingATG is underlined. Sequences in uppercase are from the parental pCGbackbone.

[0503] B. Rapamycin-Inducible Apoptosis

[0504] The ability to control Fas activation and trigger apoptosis via asmall molecule has applications both in gene therapy, where it may beused to selectively eliminate engineered cells, and in experimentalsystems. The proteins described here are anchored to the membrane viathe low affinity NGF receptor, also called p75. It should beappreciated, however, that another protein anchor could be readilysubstituted. p75 is useful experimentally because of the availability ofantibodies to its extracellular domain, and its lack of high affinityinteraction with any identified ligand (Bothwell, M. 1995. Annu. Rev.Neurosci. 18:223-253).

[0505] 1. 2-Protein Rapamycin-Regulated Fas Activation

[0506] (a) Construction of the p75 Vector

[0507] Vectors to direct the expression of FRAP-Fas fusion proteinscontaining the extracellular and transmembrane domain of the lowaffinity NGF receptor (also known as p75) were derived from themammalian expression vector pJ7W (Morgenstern, J. P. and Land, H. 1990.Nucleic Acids Res. 18:1068), modified by substitution of a pUC backbonefor the original pBR backbone using standard methods. We call thisvector pA7W. Inserts cloned into the polylinker sites of this plasmidare transcribed under the control of the simian CMV promoter andenhancer sequences. The polylinker follows the CMV sequence withHindIII-SalI-XbaI-BamHI-SmaI-Sstl-EcoRI-ClaI-KpnI-BgIII. Any mammalianexpression vector with suitable cloning sites and promoter could besubstituted.

[0508] A restriction fragment encoding a fragment of p75 flanked byHindIII and XbaI sites was generated by PCR using primers J1 (5′) and J2(3′), based on the sequence of p75 Gohnson, D., Lanahan, A., Buck, C.R., Shegal, A., Morgan, C., Mercer, E., Bothwell, M., Chao, M. 1986.Cell 47:545-554). The original source of the PCR template was a clonederived from a human brain library, using primers similar to J1 and J2but with different restriction sites. The 5′ end of the resultingfragment contains a HindIII site followed by an EcoRI site, a Kozaksequence and the initiation of p75 coding sequence (amino acid 1). The3′ end generated encodes the receptor sequence up to and including aminoacid 274, 2 amino acids past the predicted membrane spanning sequence,followed by an XbaI site. Analogous portions of other transmembranereceptors can be substituted for this fragment. The PCR product wassubcloned as a HindIII-XbaI fragment into HindIII-XbaI cut pA7W,generating pA7Wp75. The construct was verified by restriction analysisand DNA sequencing.

[0509] (b) Addition of Fas to pA7Wp75

[0510] XbaI-SpeI fragments encoding Fas amino acids 206-304 (FasS) andFas amino acids 206-319 (FasL) were made by PCR and subcloned intopA7Wp75 cut with the same enzymes. The primers used were J3 (5′) and J4or J5 (3′). J5 generates a fragment of Fas that ends beyond itstermination codon; when cut with SpeI, the nucleotides encoding theterminal 15 aa of Fas are removed to give a truncated form ofintracellular Fas we call FasS. Removal of these 15 aa increases theactivity of Fas in some cell types (Itoh, N., and Nagata, S. 1993. J.Biol. Chem 268:10932). Primer J4 replaces the natural termination codonof Fas with a SpeI site, and also mutates the original SpeI sitecontained in Fas, generating FasL. The plasmids generated fromsubcloning these fragments are pA7Wp75-FasS and pA7Wp75-FasL,respectively. These construct were verified by restriction analysis andDNA sequencing. To attach an epitope tag to these inserts, the XbaI-SpeIFas fragments were isolated and ligated into the XbaI-SpeI cut backboneof pCMF1/2/3. HA, plasmids described above which encode an epitope tagof 9 amino acids from the H. influenza haemagglutinin protein (E) 3′ tothe SpeI site, followed by a BamHI site. Cutting the resultant plasmidwith XbaI and BamHI generated fragments encoding Fas followed by theepitope tag (designated E for these constructs).

[0511] (c). p75-FRAP-Fas-Epitope Fusion Proteins: Addition ofFRAP-Containing Fragments to pA7Wp75-FasSE and pA7Wp75-FasLE to Generatep75-FRAPx-FasSorLE and p75-FasSorL-FRAPxE

[0512] The XbaI-SpeI fragments containing a portion of FRAP aredescribed previously in this document. These XbaI-SpeI fragments wereinserted into either the XbaI site directly after the p75 codingsequence to generate p75-FRAPx-FasSorLE or into the SpeI site directlyafter the Fas fragment to generate p75-FasSorL-FRAPxE. Alternatively,more than one FRAP fragment is subcloned in, either as a FRAPn fragment,or by sequential subcloning of XbaI-SpeI fragments into the SpeI siteavailable after subcloning the first FRAP into either XbaI or SpeI. Thusthe final series of vectors encodes (from the N to the C terminus) p75extracellular and transmembrane sequence, one or more FRAP-deriveddomains fused N- or C-terminally to one or more Fas intracellulardomains, and an epitope tag.

[0513] (d) p75-FKBP-Fas Fusion Proteins: Addition of FKBP-ContainingFragments to pA7Wp75-FasSE and pA7Wp75-FasLE to Generatep75-FKBPn-FasSorL or p75-FasSorL-FKBPn

[0514] The XbaI-SpeI fragments containing one or more FKBPs have beendescribed elsewhere in this document. These fragments were inserted intoeither the XbaI site directly after the p75 coding sequence to generatep75-FKBPn-FasSorL or into the SpeI site directly after the Fas fragmentto generate p75-FasSorL-FKBPn. Thus the final series of vectors encodes(from the N- to the C-terminus) p75 extracellular and transmembranesequence, one or more FKBPs fused N- or C-terminally to one or more Fasintracellular domains, and an epitope tag.

[0515] (e) Assay of Rapamycin-Mediated Fas Activation

[0516] The ability of expression of a protein containing Fas and FRAPdomains and a protein containing Fas and FKBP domains to activate Fasand trigger cell death upon addition of rapamycin can be tested ineither transiently or stably transfected cells.

[0517] For transient transfections, the two plasmids to be tested arecotransfected into a cell line such as HT1080 by a standard method suchas lipofection, calcium phosphate precipitation or electroporation. Oneor more days after transfection, cells are treated with no addition orone or more concentrations of rapamycin or one or more concentrations ofa dimerizing agent such as FK1012. The FK1012 serves as a positivecontrol that the FKBP-Fas construct is functional. Several hours to 1day later, the cells are monitored for response by one of severalmethods. Cell lysates were prepared by conventional means and used togenerate Western blots that are probed with antibody directed against HAor against the extracellular domain of p75. Alternatively, cells can beassayed by collection in isotonic solution plus 10 mM EDTA, stained withanti-p75 monoclonal antibody and labeled secondary antibody, and thepositive cells measured by FACS. A decrease in either Western blotsignal or FACS signal upon treatment indicates sucessful induction ofcell death (or decrease in protein expression). In addition,commercially available kits can be used to monitor apoptosis.

[0518] To stably transfect cells, a vector encoding a selectable markersuch as neomycin resistance is cotransfected along with the plasmidsdescribed. Two to three days after transfection, cells are plated intoG418 and the resistant population or clones are isolated by standardmeans. These populations can then be monitored directly for induction ofapoptosis by treatment with dimerizer followed by cell counting or othermeasure of cell viability.

[0519] An alternative means of generating stable cell lines expressingthe constructs of interest is to subclone the inserts into a retroviralvector. The inserts are excisable with Eco RI to facilitate thissubcloning. The vector is then used to make transducing supernatants bya packaging cell using conventional methods.

[0520] 2. Single Protein Rapamycin-Regulated Fas Activation

[0521] (a). Construction of FKBP-FRAP Chimeric Fragments FKBP-FRAPFusion Constructs for Rapamycin-Dependent Homodimerization of FasIntracellular Domain

[0522] i. Structure-Assisted Design

[0523] In order to design molecules containing both FRAP and FKBPdomains that are capable of rapamycin-dependent homodimerization, thethree dimensional structure of the ternary complex between human FKBP12,rapamycin, and a portion of human FRAP encompassing the minimal FRBdomain may be considered. Requirements for homodimerization of twomolecules of fusion proteins containing FRAP, FKBP and Fas moietiesinclude (i) sufficient length and flexibility of the polypeptide toaccomodate the distortions necessary for the FRAP-FKBP interaction tooccur between molecules tethered at the membrane, while preserving theability of aggregated Fas to transduce a signal; and (ii) prevention orminimization of intramolecular dimerization by rapamycin, an eventexpected to be highly entropically favored due to the chelate effect,and therefore to prevent the desired intermolecular moleculardimerization.

[0524] Structural considerations led us to the following designpreferences for the fusion constructs:

[0525] (i) FRB and FKBP should be joined with a polypeptide linkersufficiently short that intramolecular dimerization is stericallyprevented. The currently preferred configuration is FRAP-FKBP as theC-terminus of FRAP and the N-terminus of FKBP are distant, allowing along linker (>ten amino acids) that should still prevent intramoleculardimerization yet afford flexibility.

[0526] (ii) This FRAP-FKBP ‘cassette’ can be present membrane-proximally(i.e. with Fas domain(s) added to the C-terminus), or membrane distal(with the Fas domain membrane-proximal and the FRAP-FKBP cassetteappended C-terminally).

[0527] (iii) A long linker should be present N-terminal to the FRAP-FKBPdomains, to allow for the structural distortions implied by dimerizationat the membrane or if the domains are added C-terminally. Again aN-terminal location of FRAP is preferred as this long linker can thencomprise natural FRAP sequence from the region N-terminal to the FRBdomain, minimizing the immunogenicity of the chimeric protein.

[0528] (iv) Optimal linker lengths and fusion positions for a givenprotein should be confirmed empirically.

[0529] A series of 12 fusions of FKBP and FRAP, designated T1-T12, wasdesigned. Nine were N-FRAP-FKBP-C fusions including between 13, 23 or 33amino acids N-terminal to Arg2018 (the N-terminal linker), and 4, 7 or10 residues separating the two proteins. The remaining three wereN-FKBP-FRAP-C fusions interposing 3, 0 or −4 residues of FRAP sequencebetween FKBP Glu107 and FRAP Arg2018.

[0530] (ii) Construction

[0531] The twelve fusions were made as XbaI-BamHI cassettes that couldbe cloned directly as a single fragment, using the three-primer PCRsplicing method (Yon, J. and Fried, M. 1989. Nucleic Acids Res. 17,4895). Cloning in this way avoided the introduction of restriction sitesbetween the genes that would encode foreign sequence and alter thelength of the linker. A mixture of 1 ng each of pCGNN-1FRAPi andpCANTAB-AP-FKBP was amplified using Pfu polymerase with 1 μM each of twoouter primers (A and C), in the presence of 0.01 μM of a single ‘splice’oligo (B) complementary to both genes that directs the desired fusion.The primers used are tabulated below: N- oligos _(13 —) term* linker^(†)# construct A B C (aa) (aa) T1 FRAP(1985-2116)-FKBP 100 102 105 33 4 T2FRAP(1995-2116)-FKBP 93 102 105 23 4 T3 FRAP(2005-2116)-FKBP 101 102 10513 4 T4 FRAP(1985-2119)-FKBP 100 103 105 33 7 T5 FRAP(1995-2119)-FKBP 93103 105 23 7 T6 FRAP(2005-2119)-FKBP 101 103 105 13 7 T7FRAP(1985-2122)-FKBP 100 104 105 33 10 T8 FRAP(1995-2122)-FKBP 93 104105 23 10 T9 FRAP(2005-2122)-FKBP 101 104 105 13 10 T10FKBP-FRAP(2014-2114) 106 107 110 — 3 T11 FKBP-FRAP(2018-2114) 106 108110 — 0 T12 FKBP-FRAP(2021-2114) 106 109 110 — −4

[0532] PCR products were purified, digested with XbaI and BamHI, andligated into XbaI-BamHI digested pCM. The constructs were verified byrestriction analysis and DNA sequencing.

[0533] Primer sequences and the sequence of a representative FRB-FKBPconstruct: fusion T6 of FRAP (2005->)-FKBP are disclosed in WO 96/41865(p. 109).

[0534] (b) Addition of FRAP-FKBP Chimeric Inserts to pA7Wp75-FasSE andpA7Wp75-FasLE

[0535] Subcloning of T1 through T12 as XbaI-SpeI fragments intopA7Wp75-FasSE and pA7Wp75-FasLE linearized with XbaI generatesp75TFasSorLE. Subcloning into pA7Wp75-FasLE linearized with SpeIgenerated p75FasSorLT-E. These constructs are listed in Table 1 ((d)below).

[0536] (c) Alternative FRAP-Fas-FKBP Constructs

[0537] Instead of the format of the chimeric fragments T1-T12, thesingle chain strategy could require a different orientation of domainsfor optimal activity. To this end, another series of constructs was madein which FKBP and FRB are separated by a Fas fragment. The startingpoints for these constructs are pCMF1HA, pCMF2HA, and PCMF3HA. Similarto the strategy described above for the construction of chimerictranscription factors, FKBP and FRB fragments (described elsewhere inthis document) were cloned into the pCM backbones as XbaI-BamHIfragments that included a SpeI site just upstream of the BamHI site. AsXbaI and SpeI produce compatible ends, this allowed further XbaI-BamHIfragments to be inserted downstream of the initial insert. Additionally,cloning of an XbaI-SpeI fragment results in the addition of the fragmentat the 5′ end of the construct. The final p75-anchored construct wasmade by subcloning the XbaI-SpeI fragments shown in Table 1 ((d) below)into pA7Wp75-FasSE. A similar series is made by subcloning intopA7Wp75-FasLE. Insertion into vector cut with XbaI resulted in additionof the insert 3′ to the p75 fragment. Insertion into this vector cutwith SpeI resulted in addition of the insert 3′ to the Fas fragment.Insertion into this vector cut with XbaI and SpeI resulted in addition3′ to the p75 fragment, and elimination of the Fas fragment originallyin the vector. By using these three subcloning strategies, the followingseries of constructs was generated. Numerical subscripts define thenumber of times the domain is reiterated. TABLE 1 Code: N = p75 NGFreceptor aa 1-274 Fass = Fas aa 206-304 FasL = Fas aa 206-319 K = FKBPaa 2-108 R = FRAP 2012-2113, but other boundaries can be substituted E =HA epitope followed by termination codons as described in pCMF1/2/3.HAVECTOR SITE(S) USED TO Xba I-Spe I SUBCLONE INSERT FRAGMENT INTOpA7Wp75- NAME SUBCLONED FasSE CONSTRUCT A1 K2FasL Spe I + Xba I NK2FasLEA2 R Spe I NFasSRE A3 R Xba I NRFasSE A4 R2 Spe I NFasSR2E A5 R2 Xba INR2FasSE A6 K2FasSR Spe I NFasSK2FasSRE A7 KFasSR Spe I NFasSKFasSRE A8K2FasSR2 Spe I NFasSK2FasSR2E A9 KFasSR2 Spe I NFasSKFasSR2E A10 T1 SpeI NFasST1E A11 T2 Spe I NFasST2E A12 T3 Spe I NFasST3E A13 T4 Spe INFasST4E A14 T5 Spe I NFasST5E A15 T6 Spe I NFasST6E A16 T7 Spe INFasST7E A17 T8 Spe I NFasST8E A18 T9 Spe I NFasST9E A19 T10 Spe INFasST10E A20 T11 Spe I NFasST11E A21 T12 Spe I NFasST12E A22 K2FasSRXba I NK2FasSRFasSE A23 KFasSR Xba I NKFasSRFasSE A24 K2FasSR2 Xba INK2FasSR2FasSE A25 KFasSR2 Xba I NKFasSR2FasSE A26 T1 Xba I NT1FasSE A27T2 Xba I NT2FasSE A28 T3 Xba I NT3FasSE A29 T4 Xba I NT4FasSE A30 T5 XbaI NT5FasSE A31 T6 Xba I NT6FasSE A32 T7 Xba I NT7FasSE A33 T8 Xba INT8FasSE A34 T9 Xba I NT9FasSE A35 T10 Xba I NT10FasSE A36 T11 Xba INT11FasSE A37 T12 Xba I NT12FasSE A38 K2FasSR Spe I + Xba I NK2FasSREA39 KFasSR Spe I + Xba I NKFasSRE A40 K2FasSR2 Spe I + Xba I NK2FasSR2EA41 KFasSR2 Spe I + Xba I NKFasSR2E

[0538] (e) Termini/junction sequences of fragments, oligos and otherdetails for construction of the inserts which were cloned in 3′ to themyristoylation signal sequence as XbaI-BamHI or XbaI-SpeI fragments aredisclosed in detail in WO 96/41865.

[0539] (f) Rapamycin-Regulated Apoptosis of Stable Transsfected HumanHT1080 Cells in Culture

[0540] XbaI-BamHI fragments from constructs A30 and A31 (d, table 1)were cloned into pCM to generate M30 and M31, constructs that direct theexpression of MT5FasSE and MT6FasSE, where M denotes a myristoylationdomain (see this example sections A.1. and A.8.) and other abbreviationsare as described in d, table 1. EcoRI-BamHI fragments containing theseexpression cassettes were then cloned into the retroviral vector pSMTN3(Example 7). Helper-free retroviruses containing this DNA were generatedby transient co-transfection of 293T cells (Pear, W. S. et al. 1993.Proc. Natl. Acad. Sci. USA, 90, 8392-8396) with the constructs and aPsi(−) amphotropic packaging vector. HT1080 cells were infected withviral stock and selected with G418.

[0541] To assay apoptosis of the stably transfected pools of cells inresponse to rapamycin, cells were plated in a 96-well culture plates at10000 cells/well. After an overnight incubation, serial dilutions ofrapamycin were added, together with 50 ng/ml (final) actinomycin D, andincubation continued at 37° C. and 5% CO2 for approximately 20 hours.The media was removed and replaced with 100 μl of media containing 10%alamar blue dye. Plates were incubated as before, and the extent of cellviability assessed periodically by spectrophotometric determination ofOD at 570 nm and 600 nm on a microtiter plate reader. Typically readingwas continued until control (untreated) wells are at OD 0.2-0.4 aftersubtraction of blank. Survival of cells stably transfected with (a) M30and (b) M31-expressing constructs is potently reduced in the presence ofrapamycin, in a dose-dependent manner. The extent of cell death iscomparable to that of cells expressing a myristoylated (FKBP×2)-Fasconstruct (as disclosed in PCT/US94/08008) treated with a synthetic FKBPhomodimerizer AP1428. This system may be adapted for use with improvedrapalogs of this invention, preferably with one or more mutations in theFKBP and/or FRB domains used.

[0542] The full disclosure of each of the patent documents andscientific papers cited herein is hereby incorporated by reference.Those documents serve to illustrate the state of the art in variousaspects of this invention. Numerous modifications and variations of thepresent invention should be apparent to one of skill in the art. Suchmodifications and variations, including design choices in selecting aheterologous action domain, improved rapalog, fusion protein design, DNAformulation, viral vector or other DNA delivery means, manner and routeof transgene administration, etc. are intended to be encompassed by thescope of the invention and of the appended claims.

1 64 1 14 PRT Artificial Sequence membrane binding domain 1 Met Gly SerSer Lys Ser Lys Pro Lys Asp Pro Ser Gln Arg 1 5 10 2 4 PRT ArtificialSequence organelle binding domain 2 Lys Asp Glu Leu 1 3 4 PRT ArtificialSequence organelle binding domain 3 His Asp Glu Leu 1 4 36 DNAArtificial Sequence primer 4 gcatgtctag agagatgtgg catgaaggcc tggaag 365 35 DNA Artificial Sequence primer 5 gcatcactag tctttgagat tcgtcggaacacatg 35 6 33 DNA Artificial Sequence primer 6 gcacattcta gaattgatacgcccagaccc ttg 33 7 33 DNA Artificial Sequence primer 7 cgatcaactagtaagtgtca atttccgggg cct 33 8 36 DNA Artificial Sequence primer 8gcactatcta gactgaagaa catgtgtgag cacagc 36 9 36 DNA Artificial Sequenceprimer 9 gcactatcta gagtgagcga ggagctgatc cgagtg 36 10 36 DNA ArtificialSequence primer 10 cgatcaacta gtggaaacat attgcagctc taagga 36 11 36 DNAArtificial Sequence primer 11 cgatcaacta gttggcacag ccaattcaag gtcccg 3612 31 DNA Artificial Sequence primer 12 atgctctaga ctgggggcct tgcttggcaac 31 13 31 DNA Artificial Sequence primer 13 atgctctaga gatgagtttcccaccatggt g 31 14 39 DNA Artificial Sequence primer 14 gcatggatccgctcaactag tggagctgat ctgactcag 39 15 31 DNA Artificial Sequence primer15 atgctctaga cttggaaccg gacctgccgc c 31 16 34 DNA Artificial Sequenceprimer 16 gcatcactag tccagaaagg gcaccagcca atat 34 17 25 DNA ArtificialSequence primer 17 gccatggtgg ctagcctata gtgag 25 18 25 DNA ArtificialSequence primer 18 ggcggtgttg gctagcgtcg gtcag 25 19 27 PRT ArtificialSequence pSMTN3 construct 19 Met Ala Ser Ser Tyr Pro Tyr Asp Val Pro AspTyr Ala Ser Leu Gly 1 5 10 15 Gly Pro Ser Ser Pro Lys Lys Lys Arg LysVal 20 25 20 123 DNA Artificial Sequence pSMTN3 construct codingsequence 20 gaattccaga agcgcgtatg gcttctagct atccttatga cgtgcctgactatgccagcc 60 tgggaggacc ttctagtcct aagaagaaga gaaaggtgtc tagatatcgaggatcccaag 120 ctt 123 21 128 PRT Artificial Sequence FKBP+His+HAepitopes 21 Met His His His His His His Tyr Pro Tyr Asp Val Pro Asp TyrAla 1 5 10 15 Ala Met Ala His Met Gly Val Gln Val Glu Thr Ile Ser ProGly Asp 20 25 30 Gly Arg Thr Phe Pro Lys Arg Gly Gln Thr Cys Val Val HisTyr Thr 35 40 45 Gly Met Leu Glu Asp Gly Lys Lys Phe Asp Ser Ser Arg AspArg Asn 50 55 60 Lys Pro Phe Lys Phe Met Leu Gly Lys Gln Glu Val Ile ArgGly Trp 65 70 75 80 Glu Glu Gly Val Ala Gln Met Ser Val Gly Gln Arg AlaLys Leu Thr 85 90 95 Ile Ser Pro Asp Tyr Ala Tyr Gly Ala Thr Gly His ProGly Ile Ile 100 105 110 Pro Pro His Ala Thr Leu Val Phe Asp Val Glu LeuLeu Lys Leu Glu 115 120 125 22 27 DNA Artificial Sequence FKBP12 C24mutant primer 22 agcataaact tatggggctt gtttctg 27 23 27 DNA ArtificialSequence FKBP12 C24 mutant primer 23 agcataaact ttaagggctt gtttctg 27 2427 DNA Artificial Sequence FKBP12 C24 mutant primer 24 agcataaacttagcgggctt gtttctg 27 25 27 DNA Artificial Sequence FKBP12 C24 Mutantprimer 25 ttgcctagca tatgcttaaa gggcttg 27 26 27 DNA Artificial SequenceFKBP12 C24 mutant primer 26 ttgcctagca ttaacttaaa gggcttg 27 27 27 DNAArtificial Sequence FKBP12 C24 mutant primer 27 ttgcctagca tagccttaaagggcttg 27 28 26 DNA Artificial Sequence FKBP12 C24 mutant primer 28cctcggatca ccgcctgctt gcctag 26 29 26 DNA Artificial Sequence FKBP12 C24mutant primer 29 cagcctcgga tcgcctcctg cttgcc 26 30 30 DNA ArtificialSequence FKBP12 C13/C14 mutant primer 30 ccgggaggaa tcggctttctttccatcttc 30 31 30 DNA Artificial Sequence FKBP12 C13/C14 mutant primer31 ccgggaggaa tcgactttct ttccatcttc 30 32 30 DNA Artificial SequenceFKBP12 C13/C14 mutant primer 32 ccgggaggaa tcagatttct ttccatcttc 30 3330 DNA Artificial Sequence FKBP12 C13/C14 mutant primer 33 ccgggaggaatccattttct ttccatcttc 30 34 27 DNA Artificial Sequence FKBP12 C13/C14mutant primer 34 aagctccaca tcggcgacga gagtggc 27 35 27 DNA ArtificialSequence FKBP12 C13/C14 mutant primer 35 aagctccaca tcgccgacga gagtggc27 36 29 DNA Artificial Sequence FKBP12 C13/C14 mutant primer 36caagcatccc ggtggcgtgc accacgcag 29 37 29 DNA Artificial Sequence FKBP12C13/C14 mutant primer 37 tcccgggagg aagcaaattt ctttccatc 29 38 26 DNAArtificial Sequence FKBP12 C28/C30 mutant primer 38 cctcggatcaccgcctgctt gcctag 26 39 27 DNA Artificial Sequence FRAP C7 mutant primer39 cctttcccca aagtgcaaac gagatgc 27 40 27 DNA Artificial Sequence FRAPC7 mutant primer 40 cctttcccca aagagcaaac gagatgc 27 41 27 DNAArtificial Sequence FRAP C7 mutant primer 41 cctttcccca aaggccaaacgagatgc 27 42 29 DNA Artificial Sequence FRAp C7 mutant primer 42gttcctttcc ccatggtaca aacgagatg 29 43 29 DNA Artificial Sequence FRAP C7mutant primer 43 gttcctttcc cctaagtaca aacgagatg 29 44 29 DNA ArtificialSequence FRAP C7 muatnt primer 44 gttcctttcc ccagcgtaca aacgagatg 29 4527 DNA Artificial Sequence FRAP C7 mutant primer 45 gtcccaggcttgggcgaggt ccttgac 27 46 40 DNA Artificial Sequence FRAP C7 mutantprimer 46 gtcccaggct tggttgaggt tcgagacatt ccctgatttc 40 47 27 DNAArtificial Sequence FRAP C7 mutant primer 47 gtcccaggct tggttgaggtccttgac 27 48 29 DNA Artificial Sequence FRAP C7 mutant primer 48catgataata gagggcccag gcttgggtg 29 49 50 DNA Artificial Sequence primer49 gcatcccatg gcaatcctct ggcatgagat gtggcatgaa ggcctggaag 50 50 34 DNAArtificial Sequence primer 50 cgtgaggatc ctactttgag attcgtcgga acac 3451 48 DNA Artificial Sequence primer 51 gcatctctag aatcctctgg catgagatgtggcatgaagg cctggaag 48 52 47 DNA Artificial Sequence primer 52ggtctggatc cctaataact agtctttgag attcgtcgga acacatg 47 53 8 PRTArtificial Sequence FLAG epitope 53 Asp Tyr Lys Asp Asp Asp Asp Tyr 1 554 85 DNA Artificial Sequence oligonucleotide 54 catgtctaga gggagtagcaagagcaagcc taaggacccc agccagcgca ctagttaaga 60 attctgatga tcagcggatcctagc 85 55 85 DNA Artificial Sequence oligonucleotide 55 gctaggatccgctgatcatc agaattctta actagtgcgc tggctggggt ccttaggctt 60 gctcttgctactccctctag acatg 85 56 37 DNA Artificial Sequence oligonucleotide 56cgccttgtag aattcgcgcg tatggggagt agcaaga 37 57 30 DNA ArtificialSequence oligonucleotide 57 cccagccagc gctctagata agaattctga 30 58 18DNA Artificial Sequence oligonucleotide 58 aagggtcccc aaactcac 18 59 61DNA Artificial Sequence oligonucleotide 59 gcatgactag ttatccgtacgacgtaccag actacgcata agaaaagtga ggatcctacg 60 g 61 60 61 DNA ArtificialSequence oligonucleotide 60 ccgtaggatc ctcacttttc ttatgcgtag tctggtacgtcgtacggata actagtcatg 60 c 61 61 58 DNA Artificial Sequenceoligonucleotide 61 ccgtaggatc ctcacttttc ttaataatcg tcatcgtctttgtagtcact agtcatgc 58 62 58 DNA Artificial Sequence oligonucleotide 62gcatgactag tgactacaaa gacgatgacg attattaaga aaagtgagga tcctacgg 58 63 16PRT Artificial Sequence membrane binding domain 63 Met Gly Ser Ser LysSer Lys Pro Lys Asp Pro Ser Gln Arg Ser Arg 1 5 10 15 64 103 DNAArtificial Sequence construct containing membrane binding domainsequences 64 cgccttgtag aattcgcgcg tatggggagt agcaagagca agcctaaggaccccagccag 60 cgctctagat aagaattctg atgatcagcg gatcctgaga act 103

What is claimed:
 1. A method for multimerizing chimeric proteins incells which comprises: (a) providing cells which contain: (i) a firstrecombinant nucleic acid encoding a first chimeric protein which bindsto rapamycin or an analog thereof and which comprises at least one FKBPdomain and at least one protein domain heterologous thereto, wherein theFKBP domain comprises a peptide sequence selected from: (1) a naturallyoccuring FKBP (2) a variant of a naturally occuring FKBP in which up to10 amino acid residues have been deleted, inserted, or replaced withsubstitute amino acids, (3) an FKBP encoded by a DNA sequence capable ofselectively hybridizing to a DNA sequence encoding an FKBP of (i) or(ii); (ii) a second recombinant nucleic acid encoding a second chimericprotein which forms a complex with both (a) rapamycin or a rapamycinanalog and (b) the first chimeric protein, and which comprises at leastone FRB domain and at least one domain heterologous thereto, wherein theFRB domain comprises a peptide sequence selected from: (1) a naturallyoccuring FRB domain, (2) a variant of a naturally FRB domain in which upto 10 amino acid residues have been deleted, inserted, or replaced withsubstitute amino acids, (3) an FRB domain encoded by a DNA sequencecapable of selectively hybridizing to a DNA sequence encoding an FRB of(iv) or (v); and (b) contacting the cells with an improved rapalog whichforms a complex containing itself and at least one molecule of each ofthe first and second chimeric proteins, where the improved rapalog hasan immunosuppressive effect less than 0.01 times that of rapamycin andcomprises the substructure of formula I:

bearing one or more optional substituents, optionally unsaturated at oneor more carbon-carbon bonds spanning carbons 1 through 8, as asubstantially pure stereoisomer or mixture of stereoisomers, or apharmaceutically acceptable derivative thereof.